EP4683674A1 - Compositions and methods for delivering antibody oligonucleotide conjugates for exon skipping - Google Patents

Compositions and methods for delivering antibody oligonucleotide conjugates for exon skipping

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Publication number
EP4683674A1
EP4683674A1 EP24718015.1A EP24718015A EP4683674A1 EP 4683674 A1 EP4683674 A1 EP 4683674A1 EP 24718015 A EP24718015 A EP 24718015A EP 4683674 A1 EP4683674 A1 EP 4683674A1
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Prior art keywords
optionally substituted
seq
antibody
conjugate
antigen
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EP24718015.1A
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German (de)
French (fr)
Inventor
Elias QUIJANO
Peter Glazer
Dale Ludwig
Ravi Chari
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Yale University
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Yale University
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Publication of EP4683674A1 publication Critical patent/EP4683674A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present disclosure relates to compositions and methods for treating diseases and disorders with antibody-oligonucleotide conjugates (AOCs).
  • AOCs antibody-oligonucleotide conjugates
  • Exon skipping methodologies generally use antisense oligonucleotides (ASO) that bind splice sites in pre-mRNA for an exon containing a deleterious mutation, or bind directly to cryptic splice sites, inducing the splicing machinery to skip over the effective exon or cryptic splice site and generate a mature mRNA that lacks the affected exon, or to ignore the cryptic splice site and generate a full-length mature mRNA.
  • ASO antisense oligonucleotides
  • SMA spinal muscular atrophy
  • DMD Duchenne muscular dystrophy
  • Duchenne muscular dystrophy for example, is caused by the absence of dystrophin protein due to mutations in the dystrophin (DMD) gene.
  • the gene encoding the protein contains 79 exons spread out over more than 2 million nucleotides of DNA. Mutations disrupting the reading frame of the protein cause truncation of the translated dystrophin polypeptide, resulting in Duchenne muscular dystrophy.
  • FDA US Food and Drug Administration
  • eteplirsen Exondys 51, SEQ ID NO:208
  • Eteplirsen is an antisense oligonucleotide modified with a phosphorodiamidate morpholino oligomer (morpholino or PMO), an antisense chemistry that has been well-established in terms of its safety and effectiveness.
  • morpholino or PMO phosphorodiamidate morpholino oligomer
  • the 3E10 antibody is an ideal molecular delivery vehicle due to its efficiency in penetrating into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. 3E10 has not shown any cellular toxicity in vitro or in vivo in studies to date.
  • the present disclosure provides compositions and methods for delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to a single stranded oligonucleotide in vivo that are not reliant upon liposomal or viral vector based nucleic acid delivery.
  • the disclosure provides a conjugate of Formula (I):
  • A is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of SEQ ID NO:58, CDR2 comprising the amino acid sequence of SEQ ID NO:59, CDR3 comprising SEQ ID NO:60; and a light chain variable region (VL) CDR1 comprising the amino acid sequence of SEQ ID NO:61, CDR2 comprising the amino acid sequence of SEQ ID NO:62, CDR3 comprising the amino acid sequence of SEQ ID NO: 63;
  • VH heavy chain variable region
  • VL light chain variable region
  • L is a linker
  • P is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein; r is an integer from 1 to 4; and q is an integer from 1 to 16.
  • AA - is an amino acid sequence comprising 1 to 6 amino acid moieties.
  • each amino acid moiety of -X AA - is independently selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Vai), citrulline (Cit), and homocitrulline (HoCit).
  • -, -CR a N-. -S-, -OP(O)OR a O-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NR a -, -NR a C(O)-, -OC(O)O-, -OC(O)NR a -,
  • X 1 , X 2 , and X 3 are independently selected at each occurrence from NR a , N, CR b , S, and O.
  • the linker L is of Formula (L-l):
  • L A is a connecting moiety through which A is covalently attached to L';
  • Lp is a connecting moiety through which P is covalently attached to L'.
  • the linker L comprises at least one cleavable moiety.
  • the cleavable moiety comprises an acid-labile moiety, a reducibly-labile moiety, or an enzymatically-labile moiety.
  • the cleavable moiety comprises one or more groups selected from: wherein: each R a is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl.
  • the cleavable moiety comprises the reducibly-labile moiety -S-S-.
  • the linker L is of Formula (L-10):
  • L A is selected from a bond, -NR a '-, and -S-;
  • Lc is selected from an acid-labile moiety, a reducibly-labile moiety, and an enzymatically- labile moiety;
  • L P is selected from a bond, -NR a -, -S-, and -O-; each R a is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each R a ’ is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl; each R b is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO 3 H 2 , -PO 3 H 2 , -C(0)NR a 2 , -CO 2 R a
  • AA - is an amino acid sequence comprising 1 to 4 amino acid moieties.
  • Lc is selected from: wherein: each R a is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl.
  • Lc is -S-S-.
  • the linker L is of Formula (L-l 1):
  • L A is selected from a bond, -NH-, and -S-;
  • each Ri is independently selected from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or both Ri groups are taken together to form optionally substituted cycloalkyl; each R 2 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl;
  • AA - is an amino acid sequence comprising 2 to 4 amino acid moieties.
  • the linker L is of Formula (L-12):
  • L A is selected from a bond and -NH-;
  • L 1 ' comprises one or more groups selected from -[C(R b )2] 1-10 -, -[CH 2 CH 2 O] 1-10 -, -NR a -, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NR a -, -NR a C(O)-, -OC(O)O-, -X AA -, -OC(O)NR a -, L 2 ’ comprises one or more groups selected from -[C(R b )2] 1-10 -, -[CH 2 CH 2 O]I-IO-, -NR a -, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NR a -, -NR a C(O)-, -OC(O)O-, -X AA -, -
  • each Ri is independently selected from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 fluoroalkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or both Ri groups are taken together to form optionally substituted C 3 -C 6 cycloalkyl; each R2 is independently selected from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 fluoroalkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl;
  • -X AA - is an amino acid sequence comprising 2 or 3 amino acid moieties.
  • At least one Ri or R2 is other than hydrogen. In some embodiments, at least one Ri is an optionally substituted C 1 -C 8 alkyl. In some embodiments, each Ri is independently an optionally substituted C 1 -C 8 alkyl. In some embodiments, at least one R2 is an optionally substituted C 1 -C 8 alkyl. In some embodiments, each R2 is independently an optionally substituted C 1 -C 8 alkyl.
  • the linker L is selected from:
  • the linker is a cleavable linker.
  • the linker L is of Formula (L-20):
  • L A is selected from a bond, -NR a' -, and -S-;
  • Lp is selected from a bond, -NR a -, -S-, and -O-; each R a is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each R a is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl; and each R b is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO 3 H 2 , -PO 3 H 2 , -C(O)NR a 2 , -CO 2 R
  • the linker L is of Formula (L-21):
  • L A is selected from a bond and -NH-;
  • L P is selected from a bond and -NR a ’-; each R a is independently selected at each occurrence from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 fluoroalkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each R a is independently selected at each occurrence from hydrogen and optionally substituted C 1 -C 6 alkyl; and each R b is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO 3 H 2 , -PO 3 H 2 , -CO 2 R a , -NR a 2 , optionally substituted Ci-Cx alkyl, optionally substituted C 1 -C 8 fluoroalkyl, optionally substituted C
  • the linker L is of Formula (L-22a) or Formula (L-22b):
  • L A is selected from a bond and -NH-;
  • Lx comprises one or more groups selected from optionally substituted -[C(R b ) 2 ] 1-10 -,
  • -C C-, -CR — CR 1 -, -[CH 2 CH 2 CH 2 O] 1-10 -, -NR a -, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-,
  • Lp is selected from a bond and -NR a '-; each R a is independently selected at each occurrence from hydrogen, optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 fluoroalkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each R a ' is independently selected at each occurrence from hydrogen and optionally substituted C 1 -C 6 alkyl; and each R b is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO 3 H 2 , -PO 3 H 2 , -CO 2 R a , -NR a 2 , optionally substituted C 1 -C 8 alkyl, optionally substituted C 1 -C 8 fluoroalkyl, optional
  • the linker L is selected from:
  • the linker is a non-cleavable linker.
  • each amino acid moiety of -X AA - is independently selected from alanine (Ala), arginine (Arg), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), valine (Vai), citrulline (Cit), and homocitrulline (HoCit).
  • each amino acid moiety of -X AA - is independently selected from alanine (Ala), glycine (Gly), lysine (Lys), phenylalanine (Phe), valine (Vai), and citrulline (Cit).
  • the amino acid sequence -X AA - is selected from -Val-Cit-, -Cit-Val-, -Vai-Ala-, -Ala-Val-, -Phe-Lys-, -Lys-Phe-, -Ala-Ala-, -Val-Val-, -Gly-Gly-, -Ala-Ala-Ala-, -Gly-Gly-Gly-, Gly-Gly-Phe-Gly-(SEQ ID NO: 1032) , -Gly-Phe-Gly-Gly-(SEQ ID NO: 1033),
  • the amino acid sequence -X AA - is selected from -Val-Cit-, -Cit-Val-, -Val-Ala-, -Ala-Val-, -Phe-Lys-, -Lys-Phe-, -Ala-Ala-, -Val-Val-, -Gly-Gly-, -Ala-Ala-Ala-, and -Gly-Gly-Gly-.
  • the single stranded oligonucleotide P is a phosphorodiamidate morpholino oligonucleotide or an antisense oligonucleotide. In some embodiments, the single stranded oligonucleotide P is delivered into a muscle cell. In some embodiments, the single stranded oligonucleotide P induces skipping of exon 23 of the DMD gene.
  • the phosphorodiamidate morpholino oligonucleotide comprises the sequence 5'-C6 Amino-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO:408).
  • the antisense oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 1045, SEQ ID NOs: 158-222, SEQ ID NO:395-405, and SEQ ID NO:410-988.
  • the antisense oligonucleotide is a peptide nucleic acid (PNA) oligonucleotide.
  • the peptide nucleic acid (PNA) oligonucleotide comprises the sequence (C)-3’-TAAAGTCCATTCGGCTCCAAACCGG-C6 Amino-5’(N) (SEQ ID NO:409).
  • the single stranded oligonucleotide P comprises at least from about 10 to about 30 nucleotides in length.
  • the truncated protein modulates muscular dystrophy.
  • the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.
  • the VL CDR1 comprises the amino acid sequence of SEQ ID NO:9
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 10
  • CDR3 comprises SEQ ID NO: 11
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 15
  • CDR2 comprises the amino acid sequence of SEQ ID NO:4
  • CDR3 comprises the amino acid sequence of SEQ ID NO:5.
  • the VL CDR1 comprises the amino acid sequence of SEQ ID NO:29
  • CDR2 comprises the amino acid sequence of SEQ ID NO: 10
  • CDR3 comprises SEQ ID NO: 11
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 15
  • CDR2 comprises the amino acid sequence of SEQ ID NO:26
  • CDR3 comprises the amino acid sequence of SEQ ID NO:5.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO:21 and a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 14.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence of SEQ ID NO:20 and a full length heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 13.
  • LC full length light chain
  • HC full length heavy chain
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VL-H1 (SEQ ID NO:85), 3E10-VL-H2 (SEQ ID NO:86), 3E10-VL-H3 (SEQ ID NO:87), 3E10-VL-H4 (SEQ ID NO:88), 3E10-VL-H5 (SEQ ID NO: 89), and 3E10-VL-H6 (SEQ ID NO: 90); and a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-H1 (SEQ ID NO:64), 3E10-VH-H2 (SEQ ID NO:65), 3E1O-VH-H3 (SEQ ID NO:66), 3E10-VH-
  • VL light chain variable domain
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence selected from the group consisting of 3E10-VL-H1 (SEQ ID NO:85), 3E10-VL-H2 (SEQ ID NO:86), 3E10-VL-H3 (SEQ ID NO:87), 3E10-VL-H4 (SEQ ID NO:88), 3E10-VL-H5 (SEQ ID NO:89), and 3E10-VL-H6 (SEQ ID NO:90); and a heavy chain variable domain (VH) comprising an amino acid sequence selected from the group consisting of 3E10-VH-H1 (SEQ ID NO:64), 3E10-VH-H2 (SEQ ID NO:65), 3E10-VH-H3 (SEQ ID NO:66), 3E10-VH-H4 (SEQ ID NO:67), 3E10-VH-H5 (SEQ ID NO:68
  • the antibody or antigen-binding fragment thereof comprises a VL / VH pair selected from the group consisting of (a) VL1 (SEQ ID NO:85) and VH1 (SEQ ID NO:64), (b) VL1 (SEQ ID NO:85) and VH2 (SEQ ID NO:65), (c) VL1 (SEQ ID NO:85) and VH3 (SEQ ID NO:66), (d) VL1 (SEQ ID NO:85) and VH4 (SEQ ID NO:67), (e) VL2 (SEQ ID NO:86) and VH1 (SEQ ID NO:64), (f) VL2 (SEQ ID NO:86) and VH2 (SEQ ID NO:65), (g) VL2 (SEQ ID NO:86) and VH3 (SEQ ID NO:66), (h) VL2 (SEQ ID NO:86) and VH4 (SEQ ID NO:67), (i) VL3 (SEQ ID NO:
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising 3E10-VL-H6 (SEQ ID NOVO) and a heavy chain variable domain (VH) comprising 3E10-VH-H6 (SEQ ID NO:69).
  • VL light chain variable domain
  • VH heavy chain variable domain
  • the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising the amino acid sequence (DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLE SGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIK) (SEQ ID NO: 117) and a heavy chain variable domain (VH) comprising the amino acid sequence (EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVS S) (SEQ ID NO: 105).
  • VL light chain variable domain
  • VH heavy chain variable domain
  • the present disclosure provides a method for treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a conjugate of the disclosure to the subject.
  • the present disclosure provides a method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising administering a therapeutically effective amount of a conjugate of the disclosure to the subject.
  • DMD Duchenne muscular dystrophy
  • the disclosure provides a method for delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to a single stranded oligonucleotide to a tissue of a subject in vivo, the method including parenterally administering a pharmaceutical composition, as described herein, to the subject.
  • the antisense oligonucleotide is for treating a disease or disorder including, but not limited to a skeletal muscle disorder, a neurogenetic disease, a cardiovascular disease, a metabolic disease, or a lung disorder for which a known disease-causing mutation.
  • the 3E10 antibody or antigen-binding fragment thereof includes (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL- CDR1 (SEQ ID NO: 9), (b) a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), (c) a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRla (SEQ ID NO: 16), (e) a VH CDR2 comprising the amino acid sequence of 3E10- VH-CDR2 (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of 3E10- VH-CDR3
  • the 3E10 antibody or antigen-binding fragment thereof includes (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR1 (SEQ ID NO: 9), (b) a VL CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR2 (SEQ ID NO: 10), (c) a VL CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR3 (SEQ ID NO: 11), (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDRla (SEQ ID NO: 3), (e) a VH CDR2 comprising an amino acid sequence having no more than two amino acid
  • the 3E10 antibody or antigen-binding fragment thereof includes (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL- CDRlm (SEQ ID NO: 61), (b) a VL CDR2 comprising the amino acid sequence of 3E10-VL- CDR2m (SEQ ID NO: 62), (c) a VL CDR3 comprising the amino acid sequence of 3E10-VL- CDR3m (SEQ ID NO: 63), (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO: 58), (e) a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO: 59), and (f) a VH CDR3 comprising the amino acid sequence of
  • Figure 1 illustrates amino acid sequences for the parent 3E10 monoclonal antibody.
  • Figures 2A SEQ ID NOs: 13-25
  • 2B SEQ ID NOs:26-30
  • 2C SEQ ID NOs:31-33
  • Figures 2A illustrate amino acid sequences for the D3 IN variant ( Figure 2A), other CDR variants ( Figure 2B), and additionally contemplated CDR variants (Figure 2C) of the 3E10 monoclonal antibody, in accordance with some embodiments of the present disclosure.
  • Figure 3 illustrates example charge-conserved CDR variants of the 3E10 monoclonal antibody, in accordance with various embodiments of the present disclosure.
  • Figure 4 (SEQ ID NOs: 58-63) illustrates example CDR variants containing a combination of amino acid substitutions, charged-conserved amino acid substitutions, and rationally-designed amino acid substitutions of the 3E10 monoclonal antibody, in accordance with various embodiments of the present disclosure.
  • Figure 5 (SEQ ID NOs: 103-112) illustrates a sequence alignment of examples of humanized 3E10 heavy chain variable regions, with CDRs underlined as indicated.
  • Figure 6 illustrates a sequence alignment of examples of humanized 3E10 light chain variable regions, with CDRs and putative nuclear localization signals (NLS) underlined as indicated.
  • Figures 7A, 7B, 7C, 7D, and 7E collectively illustrate a sequence alignment of example of humanized di-scFv constructs of the 3E10 monoclonal antibody.
  • Figure 8 illustrates 3E10 (V66)-phosphorodiamidate morpholino oligomer (PMO) conjugates using three separate linker chemistries, maleimide, SATA-SAPP, and DBCO.
  • Figures 9 illustrates 3E10-phosphorodiamidate morpholino oligomer (PMO) conjugates demonstrating dose-dependent exon skipping in vitro.
  • Figure 10 illustrates single dose exon skipping in Duchenne muscular dystrophy (DMD) using a 3E10-phosphorodiamidate morpholino oligomer (PMO) conjugate. Abbreviations shown: Tibialis anterior (T), Gastrocnemius (G), Quadricep (Q), Deltoid (D), and Heart (H).
  • T Tibialis anterior
  • G Gastrocnemius
  • Q Quadricep
  • D Deltoid
  • H Heart
  • Figures 11A and 11B illustrate electrostatic surface potential renderings of a molecular model of a 3E10-scFv construct, revealing a putative Nucleic Acid Binding pocket (NAB1).
  • Figure 11A additionally shows predicted structural and electrostatic potential changes induced by amino acid substitutions at residue HC CDR1 residue 31.
  • Figure 1 IB is an illustration of molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues highlighted by punctate dots.
  • Figure 11C illustrates mapping of the putative nucleic acid binding pocket, as identified by the molecular modeling shown in Figures 11 A and 1 IB, onto the amino acid sequence of the 3E10-scFv construct.
  • Figure 12 shows the study design for confirmatory single dose exon skipping by 3E10 (V66) in mdx mice.
  • Figure 13 shows gel electrophoresis analysis of follow-on single dose IV study performed to confirm DMD functional skipping in mdx mice with 3E10 (V66) non-cleavable linker-PMO conjugate.
  • Figure 14A illustrates the results of DMD exon skipping detected across skeletal and heart muscle in mdx mice receiving non-cleavable and disulfide cleavable 3E10 (V66) PMO conjugates.
  • Figure 14B illustrates single low-dose administration of 3E10 (V66) - PMO conjugates with cleavable and non-cleavable linkers in selected tissues in mdx mice.
  • Figure 15 illustrates nucleotide sequences (SEQ ID NO: 1045, 405, and 1041-1044) for the oligomers designed to cause skipping of exon 23 in dystrophin (DMD) RNA.
  • K denotes lysine residues on PNA;
  • superscript O denotes 2’OMe modifications;
  • superscript F denotes 2’fluoro modifications;
  • superscript L denotes locked nucleic acid (LNA) modifications.
  • Figure 16 illustrates amino acid sequences of humanized 3E10 variable heavy (3E10-VH) domains (SEQ ID NOs:64-70), in accordance with various embodiments of the present disclosure.
  • Figure 17 illustrates amino acid sequences of mature humanized 3E10 heavy chains (3E10-HC) lacking a signal peptide (SEQ ID NOs:71-77), in accordance with various embodiments of the present disclosure.
  • Figure 18 illustrates amino acid sequences of humanized 3E10 heavy chains (3E10-HC) (SEQ ID NOs:78-84), in accordance with various embodiments of the present disclosure.
  • Figure 19 illustrates amino acid sequences of humanized 3E10 variable light (3E10-VL) domains (SEQ ID NOs:85-90), in accordance with various embodiments of the present disclosure.
  • Figure 20 illustrates amino acid sequences of mature humanized 3E10 light chains (3E10-LC) lacking a signal peptide (SEQ ID NOs:91-96), in accordance with various embodiments of the present disclosure.
  • Figure 21 illustrates amino acid sequences of humanized 3E10 light chains (3E10- LC) (SEQ ID NOs:97-98, 100-102, and 1045), in accordance with various embodiments of the present disclosure.
  • Figure 22 illustrates western blots of ENT2 protein expression in selected human and mouse healthy tissues.
  • Figure 23 illustrates the internalization of a labeled 3E10 (V66) - PMO conjugate in C2C12 muscle myotubes and A427 tumor cells.
  • Figure 24 illustrates the experimental design for single and repeat dose studies with 3E10 (V66) - PMO conjugates measuring exon skipping in mdx mice.
  • Figures 25A-25C illustrate the durability over time of the 3E10 (V66) - PMO conjugate administration for exon skipping in deltoid and quadriceps (Figure 25A), in tibialis anterior and gastrocnemius (Figure 25B), and in heart and diaphragm (Figure 25C).
  • Figure 26 illustrates single dose administration of the 3E10 (V66) - PMO conjugate exhibits dystrophin protein restoration.
  • Figures 27A and 27B illustrate dystrophin restoration after administration of the 3E10 (V66) - PMO conjugate in selected tissues on Day 14 ( Figure 27A) and on Day 28 ( Figure 27B).
  • Figure 28A illustrates the process for quantifying attachment sites for Lys-azide conjugation intermediates of 3E10-D31N monoclonal antibody (V66), illustrating peptides KVEPK (SEQ ID NO: 1022) and K*VEPK (SEQ ID NO: 1023) released by proteolysis.
  • Figure 28B shows the mass spectroscopy results of mapped Lys-azide conjugation intermediates of 3E10-D31N monoclonal antibody (V66) after proteolysis.
  • Figure 28C shows an alignment of chimeric 3E10-D31N variable heavy and variable light chains with the corresponding sequences of a humanized 3E10-D31N antibody (V66), in accordance with various embodiments of the present disclosure.
  • the consensus sequence for the VH is SEQ ID NO: 1024.
  • the 3E10-D31N-VH sequence is SEQ ID NO: 1025.
  • the 3E10- D31N-VH6 sequence is SEQ ID NO:69.
  • the consensus sequence for the VL is SEQ ID NO: 1026.
  • the 3E10-D31N-VL sequence is SEQ ID NO:8.
  • the 3E10-D31N-VL6 sequence is SEQ ID NO:90.
  • Figures 29A and 29B collectively illustrate improved cellular internalization of 3E10-D31N monoclonal antibody (V66) oligonucleotide conjugates in A427 cells utilizing trasglutaminase-mediated enzymatic conjugation.
  • V66 monoclonal antibody
  • Figure 30A shows improved exon skipping utilizing transglutaminase-mediated enzymatic conjugation.
  • Figure 30B shows varying the length of the PEG linkers (e.g., PEG4, PEG8, and PEG12) did not significantly impact exon skipping.
  • Figure 30C shows enhanced exon- skipping of cleavable linkers, protease Cathepsin-B and SPDMV (disulfide) compared to non- cleavable linkers with transglutaminase-mediated enzymatic conjugation.
  • PEG linkers e.g., PEG4, PEG8, and PEG12
  • SPDMV disulfide
  • Figure 31A show a di-methyl-hindered disulfide cleavable linker and protease cleavable linker have greater stability over a single methyl-hindered disulfide linker in mouse serum.
  • Figure 31B show a data table indicating the panel of antibody-oligonucleotide conjugates (AOCs) tested and their oligonucleotide to antibody ratios (OAR or DAR).
  • AOCs antibody-oligonucleotide conjugates
  • Figure 32 shows a summary table of the in vitro performance of several antibody- oligonucleotides conjugates utilizing lysine and transglutaminase-mediated conjugation.
  • Figure 33 shows levels of exon skipping of DMD preRNA at day 7 post-dose in mdx mice that were administered a 3E10 AOC with a noncleavable linker. Doses shown for each muscle group are, from left to right, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg.
  • Figure 34 shows quantification of delivered PMO at day 7 post-dose as determined by hybridization ELISA in diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker.
  • Doses shown for each muscle group are, from left to right, vehicle, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg.
  • Figure 35A shows quantification of delivered PMO at day 14 post-dose as determined by hybridization ELISA in diaphragm, quadriceps, and gastrocnemius tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker.
  • Figure 35B shows quantification of delivered PMO at day 14 post-dose as determined by hybridization ELISA in tibialis anterior, deltoid, and heart tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker.
  • Figure 36 shows levels over time of exon skipping of DMD preRNA in mdx mice that were administered a 3E10 AOC with a noncleavable linker.
  • Figure 37 shows quantification of delivered PMO at day 10 post-dose as determined by hybridization ELISA in diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker. Cohorts shown for each muscle group are, from left to right, vehicle, PMO30, and AOC30.
  • Figure 38 shows levels of exon skipping of DMD preRNA at day 10 post-dose in mdx mice that were administered a 3E10 AOC with a noncleavable linker compared to PMO alone (PMO23) or vehicle control.
  • Figure 39 shows levels of dystrophin protein restoration at day 28 post-dose in heart, diaphragm, tibialis anterior (TA), and quadriceps (Q) muscle tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker. Doses shown for each muscle group are, from left to right, 18 mg/kg, 30 mg/kg, and 42 mg/kg.
  • Figures 40A-40C show day 28 post-dose immunofluorescent detection of dystrophin in the diaphragm of WT mice ( Figure 40A), mdx mice ( Figure 40B), and mdx mice that were administered a 3E10 AOC with a noncleavable linker (Figure 40C).
  • Figure 41 illustrates a 3E10 (V66)-phosphorodiamidate morpholino oligomer (PMO) conjugate having a Cathepsin B cleavable linker.
  • Figure 42 shows levels of exon skipping of DMD preRNA at day 14 post-dose in mdx mice that were administered vehicle control, a Tg-noncleavable 3E10 AOC, and a Tg- cleavable 3E10 AOC.
  • the muscle groups shown, from left to right, are, diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H).
  • Figure 43 shows levels of exon skipping of DMD preRNA at day 14 post-dose in mdx mice that were administered vehicle control, a Lys-noncleavable 3E10 AOC, and a Tg- noncleavable 3E10 AOC.
  • the muscle groups shown, from left to right, are, diaphragm, quadriceps, gastrocnemius, tibialis anterior, deltoid, and heart.
  • Figure 44 shows levels of exon skipping of DMD preRNA at day 28 post-dose in mdx mice that were administered a noncleavable 3E10 AOC and a disulfide cleavable 3E10 AOC.
  • the muscle groups shown, from left to right, are, diaphragm, tibialis anterior (TA), heart, deltoid (Delt), gastrocnemius (Gastroc), and quadriceps (Quad).
  • Figure 45 shows day 28 post-dose levels of dystrophin protein restoration in diaphragm, tibialis anterior, quadriceps, and deltoid muscle tissue samples from mdx mice that were administered a noncleavable 3E10 AOC and a disulfide cleavable 3E10 AOC.
  • Figure 46 shows day 28 post-dose quantification of dystrophin protein distribution in diaphragm, heart, and tibialis anterior (TA) of mdx mice that were administered a noncleavable 3E10 AOC and a disulfide cleavable 3E10 AOC.
  • Figure 47 shows in situ hybridization analysis of PMO distribution in the deltoid muscle of a mouse that was administered a noncleavable 3E10 AOC, at 20x magnification.
  • Figure 48 shows in situ hybridization analysis of PMO distribution in the deltoid muscle of a mouse that was administered a noncleavable 3E10 AOC, at 40x and lOOx magnification.
  • Figure 49 shows in situ hybridization analysis of PMO distribution in the heart muscle of mice that were administered vehicle control, a cleavable 3E10 AOC, or a noncleavable 3E10 AOC, at 40x magnification.
  • Figure 50 shows quantification at day 7 post-dose of delivered PMO as determined by hybridization ELISA in deltoid, diaphragm, heart, and tibialis anterior muscle tissue samples from mice that were administered vehicle, PMO alone, PPMO alone, or a disulfide cleavable 3E10 AOC. Cohorts shown for each muscle group are, from left to right, vehicle, PMO, PPMO, and AOC.
  • Figure 51 shows in situ hybridization analysis of PMO distribution in the deltoid muscle of mice that were administered PMO alone, PPMO alone, or a disulfide cleavable 3E10 AOC, at 4x and 40x magnification.
  • Figure 52 illustrates various linker designs for generating 3E10 AOCs, i.e., a phosphatase cleavable linker, a glucuronidase cleavable linker, an SPDMV disulfide cleavable linker, and a SPDB disulfide cleavable linker.
  • Figures 53A and 53B show levels of exon skipping of DMD preRNA in mdx mice that were administered 3E10 AOCs having (Lys)-DBCO-PEG8-PMO, (Lys)-DBCO-PEG4- SPDMV-PMO, or (Lys)-DBCO-PEG8-PO4-PAB-PMO linkers ( Figure 48A), or (Tg)-DBCO- PEG8-PMO, (Tg)-DBCO-PEG4-SPDMV-PMO, or (Tg)-CathB-PEG8-PMO linkers ( Figure 48B).
  • Figure 54A illustrates the design of a site-specific DAR 4 3E10 AOC transglutaminase conjugate comprising a hindered disulfide SPDMV linker (Tg-SPDP).
  • Figure 54B shows mass spectrometry data for the site-specific DAR 4 3E10 AOC transglutaminase conjugate comprising a hindered disulfide SPDMV linker (Tg-SPDP).
  • Figure 54C shows levels of exon skipping of DMD preRNA in mdx mice that were administered a noncleavable 3E10 AOC (Tg-Peg8), or three cleavable 3E10 AOCs (Tg-SPDMV, Tg-CathB, and Tg-SPDP) .
  • compositions, conjugates, and methods for delivering therapeutic polynucleotides e g., antisense oligonucleotides
  • therapeutic polynucleotides e g., antisense oligonucleotides
  • these compositions and methods are based on the covalent attachment of a 3E10 antibody or an antigen binding fragment thereof to a single stranded oligonucleotide, forming a conjugate, increasing the in vivo effectiveness of these complexes.
  • the methods and compositions find particular use for the treatment of genetic diseases and disorders, including neurogenetic diseases, musculoskeletal disorders, cardiovascular diseases, metabolic diseases, cancers, lung disorders, and other diseases that can be benefitted by exon-skipping therapies.
  • genetic diseases and disorders including neurogenetic diseases, musculoskeletal disorders, cardiovascular diseases, metabolic diseases, cancers, lung disorders, and other diseases that can be benefitted by exon-skipping therapies.
  • compositions comprising a conjugate of (i) a 3E10 antibody or antigen-binding fragment thereof, and (ii) an antisense oligonucleotide, as well as methods for using such compositions for the treatment of diseases and disorders, including neurogenetic diseases, musculoskeletal disorders, cardiovascular diseases, metabolic diseases, cancers, lung disorders, and other diseases that can be benefitted by exon-skipping therapies described herein.
  • Example 2 demonstrates that both stable (non-cleavable) and cleavable 3E10 (V66) conjugates to a phosphorodi ami date morpholino oligomer (PMO) targeting exon 23 of DMD caused dose- dependent exon-skipping in differentiated C2C12 myotubules.
  • PMO phosphorodi ami date morpholino oligomer
  • Examples 4-6 and 9 demonstrate that V66-PMO conjugates were able to cause exon-skipping in an mdx DMD mouse model.
  • Example 7 demonstrates that there is high ENT2 protein expression in mouse and human muscle, as well as in the heart and diaphragm tissue.
  • Examples 8 and 11 demonstrate that V66-PMO conjugates are internalized in C2C12 muscle myotubes and A427 tumor cells.
  • Example 9 further demonstrates that a V66-PMO conjugate was able to restore dystrophin expression in muscle tissues in mdx mice models of DMD.
  • the advantageous properties of the compositions and methods described herein are based, at least in part, on the substantial targeting and delivery of exon-skipping oligonucleotides to muscle tissues when conjugated to 3E10 antibodies and antigen- binding fragments thereof.
  • tissue PMO delivery was observed in diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue at 7 days post- treatment.
  • the delivery demonstrated a dose-dependency in all tissues, including the heart. Delivery of the PMO exon skipping oligo to these muscle tissues was approximately 100- fold higher when conjugated to 3E10 than when injected alone without 3E10, as described in Example 14 and exemplified in Figure 37.
  • the advantageous properties of the compositions and methods described herein are based, at least in part, on the persistent localization of exon skipping oligonucleotides to when conjugated to 3E10 antibodies and antigen-binding fragments thereof.
  • tissue PMO tissue PMO localization to diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue persisted 14-days post-treatment.
  • the localization followed a dose-dependency.
  • the advantageous properties of the compositions and methods described herein are based, at least in part, on the persistent exon skipping induced by oligonucleotides when conjugated to 3E10 antibodies and antigen-binding fragments thereof.
  • exon skipping in was increased in diaphragm (Di), tibialis anterior (TA), and heart (H) muscle tissue at day 20 post-injection, relative to exon skipping at day 10 post-injection.
  • the advantageous properties of the compositions and methods described herein are based, at least in part, on the even distribution of exon-skipping oligos across muscle tissues when conjugated to 3E10 antibodies and antigen-binding fragments thereof. For instance, as described in Example 16, and exemplified in Figures 47-49, PMO oligo was evenly-distributed across deltoid and heart tissues when administered conjugated to 3E10.
  • the disclosure provides a conjugate of Formula (I):
  • the disclosure provides a method for inducing exon skipping in a target tissue in a subject by administering a conjugate of Formula (I).
  • the target tissue is a muscle tissue.
  • the target tissue is diaphragm tissue.
  • the target tissue is quadricep tissue.
  • the target tissue is gastrocnemius tissue.
  • the target tissue is tibialis anterior tissue.
  • the target tissue is deltoid tissue.
  • the target tissue is heart muscle tissue.
  • the disclosure provides a method for treating a disorder in a subject in need thereof by administering a therapeutically effective amount of a composition comprising a conjugate of Formula (I) to the subject.
  • the disorder is a muscle disorder.
  • the muscle disorder is a muscular dystrophy.
  • the muscular dystrophy is Duchenne muscular dystrophy (DMD). II. Definitions
  • antibody refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • antibody is used in the broadest sense and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and antibody fragments (such as Fab, Fab’, F(ab’)2, Fv fragments, scFv molecules), and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as they exhibit one or more of the desired biological activities.
  • “desired biological activity” of an antibody refers to the ability of the antibody to bind to its target antigen, e.g., a nucleic acid, e.g., DNA.
  • “desired biological activity” can further include antibody binding to its target antigen and resulting in a measurable biological response which can be measured in vitro or in vivo. Such activity can be antagonistic or agonistic.
  • “desired biological activity” of an antibody refers to the ability of the antibody to bind to a target, e.g., nucleic acid molecules.
  • “desired biological activity” of an antibody refers to the ability of the antibody to bind to a cellular receptor, e.g., ENT2.
  • “desired biological activity” of an antibody refers to the ability of the antibody to be internalized by a target cell.
  • Target antigen refers to the molecule that is bound specifically by the antigen-binding domain comprising the variable regions of a given antibody.
  • the term “specifically binds” refers to the binding of an antibody to its cognate antigen (e.g., a nucleic acid, e.g., DNA) while not significantly binding to other antigens.
  • antibodies can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses or isotypes, e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA . “Isotype,” as used herein, refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, y, s, y, and p. respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4 th ed. (W.B. Saunders, Co., 2000). It should be understood that antibodies disclosed herein can also comprise hybrids of isotypes and/or subclasses.
  • Antibodies of the present disclosure are generally isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. As used herein, “recombinant antibody” refers to an antibody that is generated using recombinant nucleic acid techniques in exogenous host cells, and recombinant antibodies can be isolated as well.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the CHI, CH 2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as “VH.”
  • variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies.
  • variable domains hypervariable regions
  • CDRs complementary determining regions
  • a “variable heavy domain” pairs with a “variable light domain” to form an antigen-binding domain (ABD) that specifically binds a target antigen.
  • ABS antigen-binding domain
  • the more highly conserved portions of variable domains are called the framework regions (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs/HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs/HVRs in each chain are held together in close proximity by the FR regions and, with the CDRs/HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • hypervariable region refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops.
  • antibodies comprise six HVRs or CDRs; three in the VH (Hl, H2, H3; or VH CDR1, VH CDR2, VH CDR3), and three in the VL (LI, L2, L3; or VL CDR1, VL CDR2, VL CDR3).
  • the “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“ ”), based on the amino acid sequences of their constant domains.
  • variable heavy domain (VH; containing VH CDR1, VH CDR2, and VH CDR3) and the variable light domain (VL or VL; containing the VL CDR1, VL CDR2 and VL CDR3), comprise the set of 6 CDRs, with the C-terminus of the VH domain being attached to the N-terminus of the CHI domain of the heavy chain and the C-terminus of the VL domain being attached to the N-terminus of the constant light domain (and thus forming the light chain).
  • the VH and VL domains are covalently attached, generally through the use of a linker (e.g., an “scFv linker”), into a single polypeptide sequence, which can have the N- to C-terminus arrangement of VH-linker-VL or VL- linker-VH.
  • a linker e.g., an “scFv linker”
  • the C-terminus of the scFv domain is attached to the N-terminus of the hinge in the second monomer.
  • Fab or “Fab region,” as used herein, refers to a polypeptide that comprises VH, CHI , VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g., VH- CHI on one chain and VL-CL on the other). Fab can refer to this region in isolation, or this region in the context of an antibody of the disclosure. In embodiments, a Fab comprises an Fv region in addition to CHI CL domains.
  • hinge region Another part of the heavy chain is the hinge region.
  • “hinge,” “hinge region,” “antibody hinge region,” or “hinge domain” refers to the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CHl domain ends at EU position 215, and the IgG CH 2 domain begins at residue EU position 231.
  • the antibody hinge is herein defined to include positions 216 (E216 in IgGl) to 230 (p230 in IgGl), wherein the numbering is according to the EU index as in Kabat.
  • a “hinge fragment” is used, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain.
  • Heavy chain constant region refers to the CHl-hinge-CH 2 -CH3 portion of an antibody or fragment thereof, excluding the variable heavy domain.
  • the heavy chain constant region comprises amino acids 118-447 of human IgGl, in EU numbering.
  • “heavy chain constant region fragment” refers to a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region.
  • Fv refers to a polypeptide that comprises VL and VH domains of an antibody binding domain. Fv regions can be formatted as both Fabs and scFvs, where the VL and VH domains are combined (e g., by way of a linker, as discussed herein) to form an scFv.
  • Fc refers to a polypeptide comprising CH2 -CH3 domains of an IgG molecule, and, in some cases, inclusive of the hinge.
  • the CH 2 -CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230.
  • the definition of “Fc domain” includes both amino acids 231-447 (CH 2 - CH3) and 216-447 (hinge-CH 2 -CH3) of IgGl, or fragments thereof.
  • an “Fc fragment” in this context can contain fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another Fc domain or Fc fragment as can be detected using standard methods, generally based on size (e g., non-denaturing chromatography, size exclusion chromatography, etc.).
  • the disclosed AOCs comprise human Fc domains.
  • the disclosed AOCs comprise Fc domains from human IgGl, IgG2, or IgG4.
  • variant Fc domain contains amino acid modifications as compared to a parental Fc domain.
  • a “variant human IgGl Fc domain” is one that contains amino acid modifications (generally amino acid substitutions, although in the case of ablation variants, amino acid deletions are included) as compared to the human IgGl Fc domain.
  • variant Fc domains have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding parental human IgG Fc domain.
  • the percent identity is calculated using the identity algorithms discussed below.
  • variant Fc domains have from 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid modifications as compared to the parental Fc domain.
  • variant Fc domains retain the ability to form a dimer with Ir Fc domain as measured using known techniques as described herein, such as non-denaturing gel electrophoresis.
  • EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody.
  • Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof. See, SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th edition, NIH publication, No. 91-3242, E.A.
  • amino acid position numbering is according to the IMGT system.
  • full length antibody “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
  • An “antibody fragment” comprises a portion of an intact antibody, preferably comprising the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • a “naked antibody” for the purposes herein is an antibody that is not conjugated to a payload, e.g., an oligonucleotide, cytotoxic moiety, or radiolabel.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that can be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
  • a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this disclosure.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • Antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit one or more of the desired biological activities (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or capability, while the constant regions are homologous to the sequences of antibodies derived from another species of mammals (e.g., human) to avoid eliciting an immune response In that species.
  • Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR/HVR of the recipient are replaced by residues from a CDR/HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
  • human antibody refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any technique known in the art.
  • This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide.
  • This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991).
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos.
  • a “species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species.
  • the species-dependent antibody “binds specifically” to a human antigen (e.g., has a binding affinity (Kd) value of no more than about 1 X 10 -7 M, preferably no more than about U I0 x M and preferably no more than about 1 x 10 9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen.
  • the species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
  • linear antibodies refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.
  • Modification refers to an amino acid substitution, insertion, deletion, and/or any other mutation in a polypeptide sequence.
  • ‘Variant protein,” or “protein variant,” or “variant,” as used herein refers to a protein that differs from that of a parent protein by virtue of at least one amino acid modification. The protein variant has at least one amino acid modification compared to the parent protein, yet not so many that the variant protein will not align with the parental protein using an alignment program such as that described below.
  • variant proteins are generally at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% identical to the parent polypeptide, using any alignment program known in the art, such as BLAST.
  • Sequence identity between two similar sequences can be measured by algorithms such as that of Smith, T.F. & Waterman, M.S. (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S.B. & Wunsch, CD. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol.48:443 [homology alignment algorithm], Pearson, W.R. & Lipman, D.J. (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci.
  • a parent polypeptide for example an Fc parent polypeptide, is a human wild type sequence, such as the heavy constant domain or Fc region from IgGl, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as “parent polypeptides.”
  • antibody sequences described herein have at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with a parent polypeptid
  • antibody variant refers to an antibody that differs from a parent antibody by virtue of at least one amino acid modification
  • IgG variant or variant IgG refers to an IgG that differs from a parent IgG (e.g., from a human IgG sequence) by virtue of at least one amino acid modification
  • immunoglobulin variant or variant immunoglobulin refers to an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification
  • Fc variant or “variant Fc” as used herein refers to an Fc that differs from a parent Fc, e.g., an Fc domain of human IgGl, IgG2, IgG3, or IgG4, by virtue of at least one amino acid modification.
  • IgG subclass modification or “isotype modification,” as used herein, refers to amino acid modifications that convert one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, because IgGl comprises a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an IgG subclass modification.
  • Non-naturally occurring modification as used herein is meant an amino acid modification that is not isotypic.
  • the substitution 434S in IgGl, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.
  • oligonucleotide or “polynucleotide,” used interchangeably, refers to a linear polymer of natural or modified nucleoside monomers linked by phosphodiester bonds or analogs thereof.
  • the term “oligonucleotide” usually refers to a shorter polymer, e.g., comprising from about 3 to about 100 monomers, and the term “polynucleotide” usually refers to longer polymers, e.g., comprising from about 100 monomers to many thousands of monomers, e.g., 10,000 monomers, or more. Oligonucleotides and polynucleotides can be natural or synthetic.
  • Oligonucleotides and polynucleotides can include deoxyribonucleosides, ribonucleosides, and/or non-natural analogs thereof.
  • oligonucleotides or polynucleotides are capable of specifically binding to a target genome by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • “functional nucleic acid” refers to a nucleic acid having biological functions in vivo or in cells, such as enzymatic functions, catalytic functions, or biologically inhibiting or enhancing functions (e.g., inhibition or enhancement of transcription or translation).
  • examples of functional nucleic acids include, but are not limited to, siRNA, ASO, shRNA, miRNA (including pri-miRNA and pre-miRNA), nucleic acid aptamers (including RNA aptamers and DNA aptamers), ribozymes (including deoxyribozymes), riboswitches, U1 adaptors, molecular beacons, and transcriptional factor- binding regions.
  • oligonucleotide is used in reference to an “antisense oligonucleotide.”
  • each subunit consists of: (i) a ribose sugar or a derivative thereof; and (ii) a nucleobase bound thereto, such that the order of the base-pairing moieties forms a base sequence that is complementary to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence with the proviso that either the subunit, the intersubunit linkage, or both are not naturally occurring.
  • a nucleic acid typically an RNA
  • the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In other embodiments, the antisense oligonucleotide is a 2'-O-methyl phosphorothioate (2’OMe-PS). In other embodiments, the antisense oligonucleotide is a 2’-fluoro phosphorothioate (2’F-PS).
  • the antisense oligomer of the disclosure is a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or a bridged nucleic acid (BNA) such as 2'-O,4'-C-ethylene-bridged nucleic acid (ENA).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • BNA bridged nucleic acid
  • ENA 2'-O,4'-C-ethylene-bridged nucleic acid
  • Morpholinos as described herein include all stereoisomers and tautomers of the foregoing general structure.
  • the synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063; 5,506,337; 8,076,476; and 8,299,206; all of which are incorporated herein by reference.
  • complementarity refers to two or more oligomers (i.e., each comprising a nucleobase sequence) that are related with one another by Watson-Crick base-pairing rules.
  • nucleobase sequence “T-G-A (5'— >3') is complementary to the nucleobase sequence “A-C-T (3'— >5').”
  • Complementarity may be “partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules.
  • complementarity between a given nucleobase sequence and the other nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Or, there may be “complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example.
  • the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
  • nucleobase (Nu), “base pairing moiety” or “base” are used interchangeably to refer to a purine or pyrimidine base found in naturally occurring, or “native” DNA or RNA (e.g., uracil, thymine, adenine, cytosine, and guanine), as well as analogs of these naturally occurring purines and pyrimidines. These analogs may confer improved properties, such as binding affinity, to the oligomer.
  • Exemplary analogs include hypoxanthine (the base component of inosine); 2,6-diaminopurine; 5-methyl cytosine; C5-propynyl-modified pyrimidines; 10-(9- (aminoethoxy)phenoxazinyl) (G-clamp) and the like.
  • mismatch refers to one or more nucleobases (whether contiguous or separate) in an oligomer nucleobase sequence that are not matched to a target pre- mRNA according to base pairing rules. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target pre-mRNA. Variations at any location within the oligomer are included. In certain embodiments, antisense oligomer conjugates of the disclosure include variations in nucleobase sequence near the term variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 subunits of the 5' and/or 3' terminus.
  • a “3E10 antibody” refers to an antibody with a set of heavy chain CDRs (VH CDR1, VH CDR2, and VH CDR3), identified according to the Kabat system, comprising amino acid sequences that vary from SEQ ID NOS: 58, 59, and 60 by no more than two amino acids each, respectively, a set of light chain CDRs (VL CDR1, VL CDR2, and VL CRD3) comprising amino acid sequences that vary from SEQ ID NOS: 61, 62, and 63 by no more than two amino acids each, respectively, that binds nucleic acids and is cell-penetrating at least when bound to a nucleic acid, as well as antigen-binding fragments thereof.
  • the 3E10 antigen is a polynucleotide.
  • 3E10 or “3E10 antibodies,” it will be appreciated that fragments, variants, and binding proteins, including antigen- binding fragments and fusion proteins, such as scFv, di-scFv, tr-scFv, and other single chain variable fragments, and other cell-penetrating, nucleic acid transporting molecules disclosed herein, are encompassed by the phrase and are also expressly provided for use in compositions, conjugates, and methods disclosed herein.
  • the antibodies and other binding proteins are also referred to herein as cell-penetrating.
  • the term “cell-penetrating” refers to an antibody or antigen binding fragment thereof that can penetrate a cell, e.g., a mammalian cell, without the aid of an exogeneous transport vehicle, such as a liposome, or a conjugated cell-penetrating peptide.
  • a cell e.g., a mammalian cell
  • an exogeneous transport vehicle such as a liposome
  • the cell-penetrating antibody or antigen binding fragment thereof can penetrate a cell expressing an ENT2 receptor on its cell surface in the presence of nucleic acids, e.g., non-covalently bound and/or conjugated to the 3E10 antibody or antigen binding fragment thereof, resulting in internalization of the 3E10 antibodies and antigen binding fragments thereof.
  • the cell-penetrating 3E10 antibody or antigen binding fragment thereof is conjugated to a functional molecule, e.g., a chemical agent, polynucleotide, or polypeptide.
  • a functional molecule e.g., a chemical agent, polynucleotide, or polypeptide.
  • the cell-penetrating molecules are generally referred to herein as “cell-penetrating antibodies,” it will be appreciated that fragments, including antigen-binding fragments, variants, binding proteins and fusion proteins such as scFv, di- scFv, tri-scFv, and other single chain variable fragments, and other cell-penetrating molecules disclosed herein are also expressly provided foruse in compositions, conjugates, and methods disclosed herein.
  • cell-penetrating antibodies e.g., cell-penetrating anti-DNA antibodies
  • SLE systemic lupus erythematosus
  • antibody-oligonucleotide conjugate refers to an antibody or antigen-binding fragment thereof that is covalently linked or conjugated to a biologically active molecule, for example an oligonucleotide or anti-tumor oligonucleotide, for example, an siRNA molecule, an antisense oligonucleotide.
  • a “linker” is any chemical moiety that is capable of linking or connecting a molecule, including an oligonucleotide, to a cell-binding agent such as an antibody, such as a 3E10 antibody or a fragment thereof, in a stable, covalent manner.
  • a “linker” is any chemical moiety that is capable of linking or connecting a compound such as an oligonucleotide, a polynucleotide, a DNA damage-inducing agent, a DNA repair inhibitor, an immune modulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, an adoptive cell therapy, or a topoisomerase inhibitor, to a cell-binding agent such as a 3E10 antibody or a fragment thereof, in a stable, covalent manner.
  • Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, at conditions under which the compound and/or the antibody remains active.
  • Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.
  • Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.
  • the term “subject” means any individual who is the target of administration.
  • the subject can be any animal (e.g., a mammal. Thus), including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like (e.g., which is to be the recipient of a particular treatment).
  • the subject is a human.
  • methods of the disclosure are useful in treatment a human subject.
  • the human may be referred to as a patient.
  • the human is a female.
  • the human is a male.
  • the human has an age in a range of from about 1 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
  • subject and patient as used herein include any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with an antisense oligomer conjugate of the disclosure, such as a subject (or patient) that has or is at risk for having DMD or BMD, or any of the symptoms associated with these conditions (e.g., muscle fiber loss). Also included are methods of producing dystrophin in a subject (or patient) having a mutation of the dystrophin gene that is amenable to exon 23 skipping.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphomas, Hodgkin lymphoma, Non-Hodgkin lymphoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, leukemia, neuroblastoma, Wilms tumor, bone cancer, brain stem tumor, childhood diffuse intrinsic pontine glioma (DIPG), retinoblastoma, rhabdoid tumor, sarcoma, spinal cord tumor, endocrine cancer, esophageal cancer,
  • DIPG diffuse intrinsic
  • Tumor and “neoplasm” refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous), including pre- cancerous lesions.
  • cancer cell refers to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non- tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
  • the term “pharmaceutically effective amount” means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • the precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, as well as the route of administration and the pharmacokinetics of the agent being administered.
  • carrier or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined.
  • the carrier or excipient is selected to minimize degradation of the active ingredient or to minimize adverse side effects in the subject, as would be well known to one of skill in the art.
  • the term “treat” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • antibody-oligonucleotide conjugate refers to an antibody or antigen-binding fragment thereof that is conjugated via a linker to a therapeutic oligonucleotide, for example, an oligonucleotide, an siRNA, or an antisense oligonucleotide (ASO), which may be delivered to specific cells or tissues otherwise not targetable by oligonucleotide delivery.
  • ASO antisense oligonucleotide
  • the conjugation of an oligonucleotide with an antibody or antigen- binding fragment thereof may also improve the pharmacokinetic properties of therapeutic oligonucleotides, expanding application of this therapeutic modality.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce a desired physiological effect or amount capable of achieving a desired result, particularly for treating or preventing the disorder or disease.
  • An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • an antisense oligomer administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.
  • this effect is typically brought about by inhibiting translation or natural splice-processing of a selected target sequence, or producing a clinically meaningful amount of dystrophin.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population) or the maximum tolerated dose.
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • compositions, conjugates, and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays or measurements.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model.
  • Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more.
  • Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • “enhance” or “enhancing,” or “increase” or “increasing,” or “stimulate” or “stimulating,” refers generally to the ability of one or more antisense oligomer conjugates or pharmaceutical compositions to produce or cause a greater physiological response (i.e., downstream effects) in a cell or a subject, as compared to the response caused by either no antisense oligomer conjugate or a control compound.
  • a greater physiological response may include increased expression of a functional form of a dystrophin protein, or increased dystrophin- related biological activity in muscle tissue, among other responses apparent from the understanding in the art and the description herein.
  • Increased muscle function can also be measured, including increases or improvements in muscle function by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the percentage of muscle fibers that express a functional dystrophin can also be measured, including increased dystrophin expression in about 1%, 2%, 5%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibers. For instance, it has been shown that around 40% of muscle function improvement can occur if 25-30% of fibers express dystrophin (see, e.g., DelloRusso et al, Proc Natl Acad Sci USA 99: 12979-12984, 2002).
  • An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e g., 500, 1000 times, including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) the amount produced by no antisense oligomer conjugate (the absence of an agent) or a control compound.
  • the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
  • a “functional” dystrophin protein refers generally to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy, typically as compared to the altered or “defective” form of dystrophin protein that is present in certain subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).
  • a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art.
  • dystrophin-related activity in muscle cultures in vitro can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al., Journal of Cell Science. 112:209-216, 1999).
  • Animal models are also valuable resources for studying the pathogenesis of disease, and provide a means to test dystrophin-related activity.
  • Two of the most widely used animal models for DMD research are the mdx mouse and the golden retriever muscular dystrophy (GRMD) dog, both of which are dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003).
  • dystrophin proteins can be used to measure the functional activity of various dystrophin proteins. Included are truncated forms of dystrophin, such as those forms that are produced following the administration of certain of the exon-skipping antisense oligonucleotides of the present disclosure.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • targeting sequence refers to a sequence of nucleobases of an oligomer that is complementary to a sequence of nucleotides in a target pre-mRNA.
  • the sequence of nucleotides in the target pre-mRNA is an exon 23 annealing site in the dystrophin pre-mRNA.
  • treatment of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the subject or cell.
  • Treatment includes, but is not limited to, administration of an oligomer or a pharmaceutical composition thereof, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • Treatment includes any desirable effect on the symptoms or pathology of a disease or condition associated with the dystrophin protein, as in certain forms of muscular dystrophy, and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated.
  • prophylactic treatments which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.
  • AOCs antibody-oligonucleotide conjugates
  • a cell-penetrating antibody e.g., a 3E10 antibody or antigen-binding fragment thereof
  • conjugated via a linker to an oligonucleotide, e.g., a therapeutic oligonucleotide.
  • an AOC described herein has the formula A-(L-P r ) q , wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, and P is an oligonucleotide moiety as described herein.
  • the present disclosure relates to the use of 3E10 antibodies, and derivatives thereof, for delivering antisense oligonucleotides amendable for exon skipping in tissues of a subject, including but not limited to skeletal muscle tissues for treatment of genetic skeletal muscle disorders.
  • the term antibody is used generally.
  • Antibodies that find use in the present disclosure take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments, and mimetics, described herein in various embodiments.
  • the antibody is conjugated to the biologically active molecule via a linker.
  • the antibody is a 3E10 antibody or antigen-binding fragment thereof, as described herein.
  • the antibody is a humanized 3E10 antibody or antigen-binding fragment thereof, as described herein. Any variety of agents can be transported via conjugation to the 3E10 antibody or antigen-binding fragment thereof, or humanized 3E10 antibody or antigen- binding fragment thereof, herein, such as inorganic and organic molecules, pharmaceutical agents, drugs, peptides, proteins, genetic material, and the like.
  • the antibody- oligonucleotide conjugate (AOC) comprises an oligonucleotide.
  • a . A ntigen-Binding Domains (A BDs)
  • an ABD refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope of an antigen.
  • an ABD comprises a hypervariable region, optionally a VH and/or VL domain of an antibody, optionally at least a VH domain.
  • an ABD comprises at least one complementarity determining region (CDR) of an antibody.
  • CDR complementarity determining region
  • an ABD comprises at least two CDRs of an antibody.
  • an ABD comprises at least three CDRs of an antibody.
  • an ABD comprises at least four CDRs of an antibody.
  • an ABD comprises at least five CDRs of an antibody.
  • an ABD comprises six CDRs of an antibody.
  • the present disclosure relates to the use of 3E10 antibodies and antigen binding fragments thereof, e.g., for delivering therapeutic agents (e.g., oligonucleotides) into a cell within a subject.
  • therapeutic agents e.g., oligonucleotides
  • 3E10 3E10 antibodies
  • disclosure herein referring to such antibodies also encompass antigen-binding fragments thereof, e.g., scFv, di-scFv, tr-scFv, regardless of whether it is specifically recited in each instance.
  • a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 3, 4, and 5. In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 22, 23, and 24 and VH CDRs of SEQ ID NOs: 15, 17, and 18. In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 16, 4, and 5. Other examples of 3E10 VL and VH CDR sequences are shown in Figures 1-4.
  • 3E10 is known to interact with the ENT2 nucleoside transporter expressed on various cell types, including muscle cells and cancer cells. In fact, ENT2 is overexpressed in most, if not all cancers. Accordingly, an AOC provided herein can widely target cancers based on cell surface expression of ENT2 on cancer cells. Advantageously, an AOC described herein can target ENT2 and extracellular DN A simultaneously. Importantly, 3E10 has been shown to preferentially localize into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. Targeting of the 3E10 antibody to extracellular DNA is described in, for example in Weisbart, Sci Reports, 2015, which is herein incorporated by reference. By targeting ENT2 as well as extracellular DNA, an 3E10 AOC described herein presents a platform to target a variety of cancers and deliver therapeutic oligonucleotides to target and kill cancer cells.
  • the antibody or antigen-binding fragment thereof is a murine, chimeric, humanized, or human antibody or antigen-binding fragment thereof.
  • an AOC of the present disclosure take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments, and mimetics, described herein in various embodimentspenetrates into cells and nuclei in an ENT2- dependent manner.
  • a second polynucleotide is non-covalently bound to an AOC of the present disclosure, to help facilitate cellular internalization of the AOC. That is, in some embodiments, polynucleotides conjugated to the antibody (cargo polynucleotides) do not interact with the nucleic acid-binding paratope of the antibody, and a second polynucleotide (e.g., carrier nucleic acid) is non-covalently complexed with the paratope to help facilitate internalization. In embodiments, the second polynucleotide is precomplexed with the AOC prior to administering the AOC to a subject.
  • the second polynucleotide is an extracellular polynucleotide that is bound by the AOC at a site of interest in vivo, for example, at a site of tumor ischemia and/or necrosis.
  • the second polynucleotide is DNA.
  • the second polynucleotide is RNA.
  • an AOC disclosed herein comprises a VH and VL domain of a 3E10 antibody.
  • the AOC comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a 3E10 antibody.
  • the present disclosure provides an antibody-oligonucleotide conjugate having the formula A-(L-P r )q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, and P is an oligonucleotide as described herein, wherein the linker L links A to P.
  • L is a cleavable linker and P is an ASO that mediates exon skipping.
  • the amino acid residue corresponding with D31 of the heavy chain CDR1 of the 3E10 antibody or antigen-binding fragment thereof is substituted with N.
  • 3E10 antibody variants include mutation of aspartic acid at residue 31 of VH CDR1 to arginine (3E10-D31R), which modeling indicates expands cationic charge, or lysine (3E10-D31K) which modeling indicates changes charge orientation.
  • the 3E10 antibody or antigen-binding fragment thereof includes a D31R or D3 IK substitution.
  • additional 3E10 antibody variants include R96N, and/or S30D, alone or in combination with D31N, D31R, or D31K. All of the sequences disclosed herein having the residue corresponding to 3E10 D31 or N31, are expressly disclosed with a D31R or D3 IK or N31R or N3 IK substitution.
  • the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-P r )q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is an oligonucleotide as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, wherein the linker L links A to (P); wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of XI YGMX2, where XI is D, E, N, Q, R, or K and X2 is K, R, or H (SEQ ID NO:58).
  • AOC antibody-oligonucleotide conjugate
  • the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of X1ASX2X3VSTSSYSYX4X5, where XI is K, R, or H, X2 is K, R, or H, X3 is T or S, X4 is M or L, and X5 is K, R, H, or A (SEQ ID NO:61), (b) a VL CDR2 comprising the amino acid sequence of YASYLX1S, where XI is D, E, N, or Q (SEQ ID NO:62), and (c) a VL CDR3 comprising the amino acid sequence of QX1SX2X3FPWT, where XI is K, R, or H, X2 is K, R, or H, and X3 is D or E (SEQ ID NO:63), and (d) a heavy chain variable region (VH) CDR1
  • the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-P r )q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, wherein the linker L links A to P; wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15).
  • AOC antibody-oligonucleotide conjugate
  • the antibody or antigen- binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:9), (b) a VL CDR2 comprising the amino acid sequence of YASYLES (SEQ ID NO: 10), and (c) a VL CDR3 comprising the amino acid sequence of QHSREFPWT (SEQ ID NO: 11), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15), (e) a VH CDR2 comprising the amino acid sequence of YISSGSSTIYYADTVKG (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of RGLLLDY (SEQ ID NO: 5).
  • VL light chain variable region
  • CDR complementarity determining region
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence that is identical to SEQ ID NO:21.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is identical to SEQ ID NO: 14.
  • the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence that is identical to SEQ ID NO:20.
  • the antibody or antigen-binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is identical to SEQ ID NO: 13.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:21.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 14.
  • the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:20.
  • the antibody or antigen-binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13.
  • the 3E10 antibody or antigen-binding fragment thereof can be transported into the cytoplasm and/or nucleus of the cells without the aid of a carrier or conjugate.
  • a monoclonal 3E10 antibody and active fragments thereof that are transported in vivo to the nucleus of mammalian cells without cytotoxic effect are disclosed in U.S. Patent Nos. 4,812,397 and 7,189,396 to Richard Weisbart, the disclosures of which are incorporated by reference herein, in their entireties.
  • a murine version of the 3E10 antibody is described in Zack, et al., Immunology and Cell Biology, 72:513-520 (1994), the disclosure of which is incorporated by reference herein, in its entirety.
  • Amino acid variants of the 3E10 antibody are also known in the art, for example, as described in Zack, et al., J. Immunol., 157(5):2082-8 (1996).
  • amino acid position 31, in CDR1 of the heavy chain variable region of 3E10 influences nucleic acid binding and the antibody’s ability to penetrate nuclei.
  • Substitution of the ‘wild-type’ (e.g., relative to the original murine antibody) aspartic acid by asparagine (the ‘D3 IN’ mutation) improves nucleic acid binding and nuclei penetration of the antibody, relative to the ‘wild type’ murine antibody.
  • 3E10 antibodies and antigen-binding fragments or variants thereof, with the D3 IN substitution are disclosed herein.
  • the 3E10 antibodies and antigen- binding fragments thereof disclosed herein include the D31N substitution.
  • other amino acids are substituted at position 31 in the 3E10 antibodies and antigen-binding fragments thereof disclosed herein.
  • D31R, D31K, or D31R substitutions are incorporated in some aspects of the present disclosure.
  • 3E10 light chain sequences are known in the art. See, for example, Zack, et al., J. Immunol., 15;154(4):1987-94 (1995); GenBank: L16981.1 - Mouse Ig rearranged L-chain gene, partial cds; GenBank: AAA65681.1 - immunoglobulin light chain, partial [Mus musculus]).
  • an antibody disclosed herein is an IgA, IgD, IgE, IgG, or IgM antibody, including any subtype or isotype thereof. In embodiments, an antibody disclosed herein is based on the IgG class, which has several.
  • an antibody disclosed herein is based on one of the subclasses of IgG, including, but not limited to IgGl, IgG2, IgG3, and IgG4.
  • IgGl, IgG2 and IgG4 are used more frequently than IgG3.
  • IgGl has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M), and in embodiments, antibodies disclosed herein are based on IgGl having D or E at position 356 and/or L or M at position 358.
  • the light chain generally comprises two domains, the variable light domain (containing the light chain CDRs and together with the variable heavy domains forming the Fv region), and a constant light chain region (often referred to as CL or CK).
  • the heavy chain comprises a variable heavy domain and a constant domain, which includes a CHI -optional hinge- Fc domain comprising a CH 2 -CH3.
  • the hypervariable region of an antibody generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs.
  • VH CDRs e.g. VH CDR1, VH CDR2., and VH CDR3
  • VL CDR3 e.g. VL CDR1, VL CDR2, and vlCDR3
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)).
  • the present specification uses the IMGT system to define the complementarity determining regions (CDRs) provided herein.
  • a “full CDR set” comprises the three variable light CDRs, e.g., a VL CDR1, VL CDR2, and VL CDR3, and the three variable heavy CDRs, e.g., a VH CDR1, VH CDR2, and VH CDR3. These can be part of a larger variable light or variable heavy domain, respectfully.
  • the variable heavy and variable light domains can be on separate polypeptide chains, when a heavy and light chain is used (for example when Fabs are used), or on a single polypeptide chain in the case of scFv sequences.
  • the CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies.
  • Epitope refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as nucleic acids, amino acids, or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.
  • the antibodies described herein bind to nucleic acid epitopes in a partially sequence-independent manner. That is, while the antibodies described herein bind to some polynucleotide structures and sequences with greater affinity than other nucleic acid structures and sequences, they have some general affinity for polynucleotides.
  • the “Fc domain” of the heavy chain includes the -CH 2 -CH3 domain, and optionally a hinge domain (-H-CH 2 -CH3).
  • the Fc domain comprises immunoglobulin domains CH 2 and CH3 (Cy2 and Cy3) and the lower hinge region between CHI (Cyl) and CH 2 (Cy2).
  • the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
  • “CH” domains in the context of IgG are as follows: “CHI” refers to positions 118-215 according to the EU index as in Kabat.
  • the “Fc domain” includes the -CH 2 -CH3 domain, and optionally a hinge domain (hinge-CH 2 -CH3).
  • a scFv when attached to an Fc domain, it is generally the C-terminus of the scFv construct that is attached to all or part of the hinge of the Fc domain; for example, it is generally attached to the sequence EPKS which is the beginning of the hinge.
  • amino acid modifications are made to the Fc region, for example to alter binding to one or more FcyR receptors or to the FcRn receptor, and to enable heterodimer formation and purification, as outlined herein.
  • the hinge region is another part of the heavy chain.
  • hinge region or “hinge region” or “antibody hinge region” or “hinge domain” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.
  • the present disclosure refers to different antibody domains of a 3E10 antibody or antigen- binding fragment thereof.
  • the IgG CHI domain ends at EU position 215, and the IgG CH 2 domain begins at residue EU position 231.
  • the antibody hinge is herein defined to include positions 216 (E216 in IgGl) to 230 (p230 in IgGl), wherein the numbering is according to the EU index as in Kabat.
  • a “hinge fragment” is used, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain.
  • a scFv comprises a variable heavy chain, an scFv linker, and a variable light domain.
  • the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable light chain (N-vh-linker-vl-C) although that can be switched (N- vl-linker-vh-C).
  • the present disclosure relates to different antibody domains.
  • These domains include, but are not limited to, the Fc domain, the CHI domain, the CH 2 domain, the CH3 domain, the hinge domain, the heavy constant domain (CHl-hinge-Fc domain or CHl-hinge-CH 2 -CH3), the variable heavy (VH) domain, the variable light (VL) domain, the light constant domain, Fab domains and scFv domains.
  • the antibodies of the disclosure comprise a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.
  • such an antibody comprises or consists of a murine, chimeric, humanized, or human antibody or antigen-binding fragment thereof comprising heavy or light chain variable regions that are "“the product of'” or ““derived from”” a particular germline sequence, e.g., that of the 3E10 antibody.
  • a human antibody that is "“the product of 1 ” or ““derived from”” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody (using the methods outlined herein).
  • a human antibody that is “the product of’ or “derived from” a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation.
  • a humanized antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as being derived from human sequences when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences).
  • a humanized antibody is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a humanized antibody derived from a particular human germline sequence will display no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene.
  • the humanized antibody has no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
  • a parental antibody is affinity matured.
  • Methods for affinity maturation are known in the art.
  • structure-based methods are employed for humanization and affinity maturation, for example, as described in U.S Patent Publication No. 2006/0008883, which is incorporated herein by reference.
  • Selection based methods are also known for humanization and/or affinity maturation of antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272(16): 10678-10684; Rosok et al., 1996, J. Biol. Chem.
  • one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • the Fc region variant can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • an Fc region variant possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays known in the art can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • a CDC assay can be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006)).
  • an antibody provided herein can have reduced effector function and thus can comprise a substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
  • an Fc region variant provided herein can have improved or diminished binding to FcRs. See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001), the disclosure of which are incorporated herein by reference, in their entireties.
  • an Fc region variant provided herein comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • an Fc region variant provided herein comprises alterations that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • an Fc region variant provided herein comprises alterations that result in increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), e.g., as described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • FcRn neonatal Fc receptor
  • Such Fc variants include those with substitutions at one or more ofFc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
  • an Fc region variant provided herein comprises “knob-in-hole” or “skew” variants, which refer to amino acid engineering that creates stearic influences to favor heterodimeric formation and disfavor homodimeric formation, as described in USSN 61/596,846, Ridgway et al, Protein Engineering 9(7):617 (1996); Atwell et al, J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, all of which are hereby incorporated by reference in their entirety.
  • an Fc region variant provided herein comprises alterations described in Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351.
  • the antibody portion of an AOC described herein comprises an antigen-binding fragment of a 3E10 antibody.
  • the antigen-binding fragment retains the desired biological activity of a 3E10 antibody.
  • the antigen-binding fragment retains at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the desired biological activity of a 3E10 antibody.
  • the antigen-binding fragment retains the ability of the antibody to bind to its target antigen, e.g., a nucleic acid, e.g., DNA. In embodiments, the antigen-binding fragment retains the ability of the antibody to bind to a cellular receptor, e.g., ENT2. In embodiments, the antigen- binding fragment retains the ability of the antibody to be internalized by a target cell.
  • the 3E10 antibody or antigen binding fragment thereof comprises a single-chain fragment variable (scFv), a tandem double scFv, an (scFv)2, a minibody, a VHH, an scFv-Fc, a CrossMab, a dual variable domain immunoglobulin (DVD-Ig), a single-chain tandem fragment variable (scTaFv), a diabody, a tandem diabody (TandAb), a Fabsc, a modular IgG-scFv, a Fab, or an F(ab’)2.
  • scFv single-chain fragment variable
  • scFv tandem double scFv
  • an (scFv)2 a minibody
  • VHH an scFv-Fc
  • CrossMab a dual variable domain immunoglobulin
  • DVD-Ig dual variable domain immunoglobulin
  • scTaFv single-chain tandem fragment variable
  • diabody
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a CrossMab.
  • CrossMab complementary mutations are introduced in the heavy chain constant region of each arm to generate so-called “holes and knobs,” resulting in preferred association between different arms, forming a heterodimer, rather than a homodimer of two of the same arms.
  • the exact residues that are mutated in the heavy chain constant region of a CrossMab bispecific antibody to form “holes” and “knobs” can vary depending on the specific design and optimization goals of the antibody.
  • CrossMab antibodies see, for example, Huang, J., et al., Journal of Biological Chemistry, 294(50): 19001-10 (2019), the disclosure of which is incorporated herein by reference in its entirety.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a divalent, dual-variable domain immunoglobulin (DVD-Ig) of a 3E10 antibody or antigen binding fragment thereof.
  • DVD-Ig divalent, dual-variable domain immunoglobulin
  • each arm of the antibody contains two VH/VL pairs.
  • one of the VH/VL pairs comprises 3E10 VH and VL CDRs.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a single-chain variable fragment (scFv).
  • Single chain Fv or “scFv,” as used herein, refers to a VH domain covalently attached to a VL domain through a linker, e g., a scFv linker as discussed herein, to form a continuous protein chain.
  • a scFv domain can be in either arrangement from N- to C-terminus (i.e., VH-linker-VL or VL-linker-VH).
  • H.X L.Y means the N- to C-terminus arrangement is VH- linker-VL
  • L.Y H.X means the N- to C-terminus arrangement is VL-linker-VH.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a tandem double scFv.
  • a tandem double scFv has two scFv domains linked in a linear fashion.
  • each scFv domain is derived from a different antibody and provides independent antigen-binding specificity.
  • one of the scFv domains comprises 3E10 VH and VL CDRs.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a dimeric scFv antibody (scFv)2.
  • a dimeric scFv antibody has two scFv domains linked in a dimeric arrangement.
  • each scFv domain is derived from a different antibody and provides independent antigen-binding specificity.
  • one of the scFv domains comprises 3E10 VH and VL CDRs.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a scFv-Fc.
  • An “scFv-Fc,” as meant herein, is a polypeptide that consists of a heavy and a light chain variable region of an antibody joined by a linker, which is followed by an Fc polypeptide chain of an antibody, optionally the Fc region of a human IgG antibody, such as an IgGl, IgG2, IgG3, or IgG4 antibody.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a single-chain tandem fragment variable (scTaFv) antibody.
  • a single-chain tandem fragment variable (scTaFv) antibody is a type of bispecific antibody that consists of two variable fragment (VH and VL) domains linked in a tandem arrangement.
  • one of the variable fragment domains comprises 3E10 VH and VL CDRs.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a VHH, also referred to as a nanobody.
  • VHH refers to a variable domain of heavy chain of heavy-chain antibody.
  • a VHH is a molecule that can recognize an antigen through a single domain and is the smallest unit among antibody molecules that have been found to date.
  • a VHH can include one or more variable domains of heavy chain derived from a heavy-chain antibody, and the number of the variable domains of heavy chain included in the VHH is not limited.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a diabody.
  • diabody refers to a divalent antibody comprising two polypeptide chains, wherein each polypeptide chain is too short for a pair to form between two domains on the same chain such that each domain is paired with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-48 and Poljak et al., 1994, Structure 2: 1121-23).
  • one of the antigen binding domains of the diabody comprises 3E10 VH and VL CDRs.
  • Polypeptide chains of different sequences can be used to prepare diabodies with two different antigen-binding sites.
  • triabodies and tetrabodies refer to antibodies that contain three and four polypeptide chains, respectively, and form three and four antigen-binding sites (which can be the same or different), respectively.
  • minibody is used to refer to an scFv-CH3 fusion protein that self- assembles into a bivalent dimer of 80 kDa (ScFv-CH3)2.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a tandem diabody (TandAb).
  • a tandem diabody has two antigen-binding domains (VH and VL) linked in a tandem arrangement by a flexible peptide linker.
  • one of the antigen binding domains comprises 3E10 VH and VL CDRs.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a Fabsc.
  • a “Fabsc” format antibody molecule typically refers to a bispecific antibody molecule having a Fab fragment, which generally includes a hinge region, which is at the C-terminus of the Fab fragment linked to the N- terminus of a CH 2 domain, of which the C-terminus is in turn linked to the N-terminus of a scFv fragment.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a scFab.
  • a scFab also known as a single-chain fragment antigen binding (Fab) is a type of antibody fragment that combines the variable heavy chain (VH) and variable light chain (VL) domains into a single polypeptide chain, linked by a peptide linker.
  • the domain structure of a Fabsc includes the variable domains of both the heavy chain and light chain (VH and VL), and a peptide linker that connects the two domains.
  • a Fabsc also includes the constant domains of the light chain (CL) and the hinge region of the heavy chain.
  • one of the antigen binding domains comprises 3E10 VH and VL CDRs.
  • Fabscs see, for example, Kettner, C., et al., Frontiers in Immunology, 8(8):453 (2017), the disclosure of which is incorporated herein by reference in its entirety.
  • an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises an IgG-scFv.
  • An IgG-scFv is an antibody in which a scFv is fused to the light chain or heavy chain of an IgG.
  • the scFv comprises 3E10 VH and VL CDRs.
  • the IgG comprises 3E10 VH and V LCDRs.
  • the antibody is an F(ab’)2.
  • 3E10 antibodies and antigen-binding fragments thereof can be modified to improve their therapeutic potential.
  • the cell-penetrating anti-DNA antibody is conjugated to another antibody specific for a second therapeutic target in the cytoplasm and/or nucleus of a target cell.
  • the cell-penetrating 3E10 antibody is a bispecific antibody having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds a second therapeutic target.
  • Bispecific antibodies and other binding proteins having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds a second target are discussed in Weisbart, et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart, et al., Int. J. Oncology, 25: 1113-8 (2004), and U.S. Patent Application No. 2013/0266570, which are specifically incorporated by reference in their entireties.
  • the second target is specific for a target cell-type, tissue, organ, etc.
  • the second heavy chain and second light chain can serve as a targeting moiety that targets the complex to the target cell-type, tissue, organ.
  • the second heavy chain and second light chain target, hematopoietic stem cells, CD34 + cells, T cells or any another cell type of interest, e g., by targeting a receptor or ligand expressed on the cell type of interest.
  • the second heavy chain and second light chain target the thymus, spleen, or cancer cells.
  • Bispecific antibodies can be used to direct cytotoxic agents or drugs to cells which express a particular antigen. These antibodies possess two binding sites directed at two different antigens or two different epitopes on the same antigen.
  • thebispecific can comprise one arm for ENT2 engagement and another arm for a second target.
  • Bispecific antibody design can include a variety of antibody designs with multiple binding arms. Techniques for making bispecific antibodies are common in the art (Millstein et al., 1983, Nature 305:537- 539; Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121 : 120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol.
  • Antibodies with more than two valencies are also contemplated.
  • trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60 (1991)).
  • the contemplated bispecific antibody disclosed herein can be conjugated as a bispecific antibody-payload conjugate.
  • Heteroconjugate antibodies are also within the scope of the present disclosure.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate.
  • the contemplated herteoconjugate antibody disclosed herein can be conjugated as a heteroconjugate antibody-payload conjugate.
  • a 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences corresponding to the parent 3E10 antibody.
  • a 3E10 antibody or antigen-binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of X1ASX2X3VSTSSYSYX4X5, where XI is K, R, or H, X2 is K, R, or H, X3 is T or S, X4 is M or L, and X5 is K, R, H, or A (SEQ ID NO:61), (b) a VL CDR2 comprising the amino acid sequence of YASYLX1S, where XI is D, E, N, or Q (SEQ ID NO:62), and (c) a VL CDR3 comprising the amino acid sequence of QX1 SX2X3FPWT, where XI is K, R, or H, X2 is K, R, or H, and X3 is D or E (SEQ ID NO:63), and (d) a
  • a 3E10 antibody or antigen-binding fragment thereof includes a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDR1 (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).
  • VL light chain variable region
  • CDR complementarity determining region
  • a 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences from a variant 3E10 antibody that includes a D31N amino acid substitution in the VH CDR1.
  • the a 3E10 antibody or antigen- binding fragment thereof includes a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 D3 IN (SEQ ID NO:22), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 D31N (SEQ ID NO:23), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 D31N (SEQ ID NO:24), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH- CDR1 D31N (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence of 3E10-
  • a 3E10 antibody or antigen-binding fragment thereof described herein refers to CDR sequences corresponding to the parent 3E10 antibody, optionally including a D3 IN amino acid substitution in the VH CDR1.
  • a 3E10 antibody or antigen-binding fragment thereof includes a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (VL) complementarity determining region (CDR
  • a 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences corresponding to the parent 3E10 antibody, with a known amino acid substitution in one or more CDR. Accordingly, in embodiments, a 3E10 antibody or antigen- binding fragment thereof described herein includes one or more amino acid substitution, relative to the CDR sequences of the parent 3E10 or 3E10-D31N variant, selected from a G to S substitution at position 5 of VH CDR2, a T to S substitution at position 14 of VH CDR2, an S to T substitution at position 5 of VL CDR1, an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.1 (SEQ ID NO:26) or 3E10-VH-CDR2.2 (SEQ ID NO:27).
  • the 3E10 antibody or antigen- binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.1 (SEQ ID NO:28) or 3E10-VL- CDR1.2 (SEQ ID NO:29).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 according to the 3E10- D3 IN variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.1 (SEQ ID NO:30). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.3 (SEQ ID NO:32). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant, e g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant, e g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDRl.cl (SEQ ID NO:34), 3E10-VH- CDRl.c2 (SEQ ID NO:35), 3E10-VH-CDRl.c3 (SEQ ID NO:36), 3E10-VH-CDRl.c4 (SEQ ID NO:37), or 3E10-VH-CDRl.c5 (SEQ ID NO:38).
  • the 3E10 antibody or antigen- binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 2 and 3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 2 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.cl (SEQ ID NO:39), 3E10-VH- CDR2.c2 (SEQ ID NO:40), or 3E10-VH-CDR2.c3 (SEQ ID NO:41).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 -3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3.cl (SEQ ID NO:42), 3E10-VH- CDR3.c2 (SEQ ID NO:43), or 3E10-VH-CDR3.c3 (SEQ ID NO:44).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 -3, and VH CDRs 1 and 2 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.C1 (SEQ ID NO:45), 3E10-VL- CDR1.C2 (SEQ ID NO:46), 3E10-VL-CDRl.c3 (SEQ ID NO:47), 3E10-VL-CDRl.c4 (SEQ ID NO:48), 3E10-VL-CDR1.C5 (SEQ ID NO:49), or 3E10-VL-CDRl.c6 (SEQ ID NO:50).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.cl (SEQ ID NO:51).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1- 3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3.cl (SEQ ID NO:52), 3E10-VL- CDR3.C2 (SEQ ID NO:53), 3E10-VL-CDR3.c3 (SEQ ID NO:54), 3E10-VL-CDR3.c4 (SEQ ID NO:55), 3E10-VL-CDR3.c5 (SEQ ID NO:56), or 3E10-VL-CDR3.c6 (SEQ ID NO:57).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1- 3 according to the 3E10- D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes any combination of the 3E10 CDR amino acid substitutions described above.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO:58).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 2 and 3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO:59).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO:60).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the 3E10-D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO:61). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO:62).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1 - 3 according to the 3E10-D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO:63).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1-3 according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1- 3 according to the 3E10-D31N variant.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
  • a 3E10 antibody or antigen-binding fragment thereof described herein includes a VL CDR 1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO:61), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO:62), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO:63), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO:58), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO:59), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO:60).
  • VL CDR 1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO:
  • a 3E10 antibody or antigen-binding fragment thereof described herein refers to CDR sequences having no more than one amino acid substitution relative to the parent 3E10 antibody optionally including a D31N amino acid substitution in the VH CDR1.
  • a 3E10 antibody or antigen-binding fragment thereof includes a VL CDR 1 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDR2 (SEQ ID NO:4), and a VH CDR3 comprising an amino acid sequence having no more than one amino acid substitution
  • a 3E10 antibody or antigen-binding fragment thereof described herein refers to CDR sequences having no more than two amino acid substitution relative to the parent 3E10 antibody optionally including a D31N amino acid substitution in the VH CDR1.
  • a 3E10 antibody or antigen-binding fragment thereof includes a VL CDR 1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR2 (SEQ ID NON), and a VH CDR3 comprising an amino acid sequence having no more than
  • 3E10 antibody or antigen-binding fragment thereof are also known in the art, as disclosed for example, in Zack, et al., J. Immunol., 157(5):2082-8 (1996).
  • amino acid position 31 of the heavy chain variable region of 3E10 has been determined to be influential in the ability of the antibody and fragments thereof to penetrate nuclei and bind to DNA.
  • a D31N mutation in CDR1 penetrates nuclei and binds DNA with much greater efficiency than the original antibody (Zack, et al., Immunology and Cell Biology, 72:513-520 (1994), Weisbart, et al., J. Autoimmun., 11, 539-546 (1998); Weisbart, Int. J. Oncol., 25, 1867-1873 (2004)).
  • the antibody or antigen-binding fragment described herein has the D3 IN substitution.
  • Antibody-payload conjugates described herein can be prepared with any 3E10 antibodies or antigen-fragments thereof, or any humanized 3E10 antibodies or antigen-fragments thereof, disclosed in the prior art. See, for example WO 2015/106290, 2016/033324, WO 2019/018426, and WO 2019/018428 (each of which is specifically incorporated by reference herein, in its entirety).
  • an antibody -payload conjugate provided herein comprises a humanized 3E10 antibody.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • the disclosure provides humanized antibodies, or antigen-binding fragments thereof, that incorporate any combination of the humanized VL and VH sequences disclosed here, as well as VL and VH sequences having sequence identity thereto, e.g., having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a VH or VL sequence described herein.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of any one of SEQ ID NOs: l, 13, or 71-84.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 1.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 13.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:71.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:72.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:73.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:74.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:75.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:76.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:77.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:78.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:79.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:80.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:81.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 82.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:83.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 84.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of any one of SEQ ID NOs:2, 14, 64-70, 103-112.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:2.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 14.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 64. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 65. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:66.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:67. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:68. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:69.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:70. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 103. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 104.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 105.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 106.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 107.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 108. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 109. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 110.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 111.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 112.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of any one of SEQ ID NOs:7, 20, or 91-102.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:7.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:20.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:91.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:92.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:93.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:94.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:95.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:96.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:97.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:98.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:99.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO: 100.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO: 101.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO: 102.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of any one of SEQ ID NOs:8, 21, 85-90, or 113-121.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 8.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:21.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:85. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 86. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:87.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:88. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:89. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NOVO.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 113. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 114. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 115.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 116.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 117.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 118.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 119. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 120. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 121.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of any one of SEQ ID NOs: 122-137.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 122.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 123.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 124.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 125.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 126.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 127.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 128.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 129.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 130.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 131.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 132.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 133.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 134.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 135.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO:136.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 137.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: l, 13, or 71-84.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 13.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:71.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:72.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:73.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:74.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:75.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:76.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:77.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%>, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:78.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%) identity to SEQ ID NO:79.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%>, at least about 80%>, at least about 85%>, at least about 90%>, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:80.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%) identity to SEQ ID NO:81.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%o, at least about 98%>, at least about 99%, or 100%) identity to SEQ ID NO:82.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:83.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%>, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:84.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs:7, 20, or 91-102.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:7.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:20.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:91.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:92.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:93.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:94.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:95.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:96.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:97.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:98.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:99.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 100.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 101.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 102.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 2, 14, 64-70, or 103-112.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:2.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 14.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:64.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:65.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:66.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:67.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:68.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:69.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:70.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 103.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 104.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 105.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 106.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 107.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 108.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 109.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 110.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:111.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 112.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 8, 21, 85-90, or 113-121.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:8.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:21.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:85.
  • an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 86.
  • an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:87.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:88.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:89.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NOVO.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 113.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 114.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 115.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 116.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 117.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 118.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:119.
  • an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 120.
  • an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 121.
  • an antibody -payload conjugate provided herein comprises a humanized 3E10 antibody, or antigen-binding fragment thereof, comprising a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least about 97% identical to an amino acid sequence selected from the group consisting of 3E10-VL-hl (SEQ ID NO:85), 3E10- VL-h2 (SEQ ID NO:86), 3E10-VL-h3 (SEQ ID NO:87), 3E10-VL-h4 (SEQ ID NO:88), 3E10- VL-h5 (SEQ ID NO:89), and 3E10-VL-h6 (SEQ ID NOVO) and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-hl (SEQ ID NO:64), 3E10-VH-h2 (SEQ ID NO:65),
  • the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-hl (SEQ ID NO:85). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-hl (SEQ ID NO:85). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-hl (SEQ ID NO:85). In embodiments, the sequence of the 3E10-VL is 3E10-VL-hl (SEQ ID NO:85).
  • the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h2 (SEQ ID NO:86). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h2 (SEQ ID NO:86). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h2 (SEQ ID NO:86). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h2 (SEQ ID NO:86).
  • the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h3 (SEQ ID NO:87). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h3 (SEQ ID NO:87). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h3 (SEQ ID NO:87). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h3 (SEQ ID NO:87).
  • the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h4 (SEQ ID NO:88). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h4 (SEQ ID NO:88). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h4 (SEQ ID NO:88). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h4 (SEQ ID NO:88).
  • the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h5 (SEQ ID NO:89). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h5 (SEQ ID NO:89). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h5 (SEQ ID NO:89). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h5 (SEQ ID NO:89).
  • the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h6 (SEQ ID NO:90). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h6 (SEQ ID NO:90). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h6 (SEQ ID NO:90). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h6 (SEQ ID NO:90).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-hl (SEQ ID NO:64).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is 3E10-VH-hl (SEQ ID NO:64).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h2 (SEQ ID NO:65).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h2 (SEQ ID NO:65).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h3 (SEQ ID NO:66).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h3 (SEQ ID NO:66).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h4 (SEQ ID NO:67).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h4 (SEQ ID NO:67).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h5 (SEQ ID NO:68).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h5 (SEQ ID NO:68).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h6 (SEQ ID NO:69).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h6 (SEQ ID NO:69).
  • the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h7 (SEQ ID NO:70).
  • sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h7 (SEQ ID NO:70).
  • an antibody -payload conjugate comprising a humanized 3E10 antibody, or antigen-binding fragment thereof, described herein includes a light chain (3E10-LC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-LC-hlm (SEQ ID NO:91), 3E10-LC-h2m (SEQ ID NO:92), 3E10-LC-h3m (SEQ ID NO:93), 3E10-LC-h4m (SEQ ID NO:94), 3E10-LC-h5m (SEQ ID NO:95), and 3E10-LC-h6m (SEQ ID NO:96) and a heavy chain (3E10-HC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-hlm (SEQ ID NO:71), 3E10-HC-h2m (SEQ ID NO: 72), 3E10-LC-hlm
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-hlm (SEQ IDN0:91).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is 3E10-LC-hlm (SEQ ID NO:91).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h2m (SEQ IDNO:92).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h2m (SEQ ID NO:92).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h3m (SEQ ID NO:93).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h3m (SEQ ID NO:93).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h4m (SEQ ID NO:94).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h4m (SEQ ID NO:94).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h5m (SEQ ID NO:95).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h5m (SEQ ID NO:95).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h6m (SEQ IDNO:96).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h6m (SEQ ID NO:96).
  • sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-hlm (SEQ ID NO:71).
  • sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is 3E10-HC-hlm (SEQ ID NO:71).
  • sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h2m (SEQ ID NO:72).
  • the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h2m (SEQ ID NO:72). [0314] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h3m (SEQ ID NO:73).
  • sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h3m (SEQ ID NO:73).
  • sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h4m (SEQ ID NO:74).
  • sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h4m (SEQ ID NO:74).
  • sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h5m (SEQ ID NO:75).
  • sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h5m (SEQ ID NO:75).
  • sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h6m (SEQ ID NO:76).
  • sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h6m (SEQ ID NO:76).
  • sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h7m (SEQ ID NO:77).
  • sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h7m (SEQ ID NO:77).
  • an antibody-payload conjugate provided herein comprises a humanized 3E10 antibody, or antigen-binding fragment thereof, comprising a light chain (3E10- LC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-LC-hl (SEQ ID NO:97), 3E10-LC-h2 (SEQ ID NO:98), 3E10-LC-h3 (SEQ ID NO:99), 3E10-LC-h4 (SEQ ID NO: 100), 3E10-LC-h5 (SEQ ID NO: 101), and 3E10-LC-h6 (SEQ ID NO: 102) and a heavy chain (3E10-HC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-hl (SEQ ID NO:78), 3E10-HC-h2 (SEQ ID NO:79), 3E10-HC- h3 (SEQ ID NO:
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-hl (SEQ ID NO:97. In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is 3E10-LC-hl (SEQ ID NO:97).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h2 (SEQ ID NO:98).
  • the sequence of the 3E10-LC is 3E10-LC-h2 (SEQ ID NO:98). [0322] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h3 (SEQ ID NO:99).
  • sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h3 (SEQ ID NO:99).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h4 (SEQ ID NO: 100).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h5 (SEQ ID NO: 101).
  • the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h6 (SEQ ID NO:102). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h6 (SEQ ID NO: 102).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is 3E10-HC-hl (SEQ ID NO:78).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h2 (SEQ ID NO:79).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-113 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h3 (SEQ ID NO:80).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h4 (SEQ ID NO:81).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h5 (SEQ ID NO:82).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h6 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h6 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-116 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h6 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h6 (SEQ ID NO:83). In some aspects, the sequence of the 3E10-HC is 3E10-HC-h6 (SEQ ID NO:83).
  • the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h7 (SEQ ID NO: 84). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h7 (SEQ ID NO:84).
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein has CDR sequences corresponding to those in the parent 3E10 antibody, optionally including a D31N amino acid substitution in the VH CDR1.
  • a humanized 3E10 antibody or antigen-binding fragment thereof includes a light chain variable domain (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable domain (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).
  • VL light chain variable domain
  • CDR complementarity determining region
  • an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences from a variant humanized 3E10 antibody that includes a D31N amino acid substitution in the VH CDR1 (SEQ ID NO: 15).
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than seven amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than ten amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10- VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NON 1), 3E10-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
  • CDRs complementarity determining regions
  • an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than nine amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
  • CDRs complementarity determining regions
  • an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than eight amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than seven amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO:11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than six amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10- VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NO:11), 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than five amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
  • CDRs complementarity determining regions
  • an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than four amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
  • CDRs complementarity determining regions
  • an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than three amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than two amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO:11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than one amino acid substitution, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10- VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NO:11), 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
  • CDRs complementarity determining regions
  • an antibody-payload conjugate described herein can comprise a humanized 3E10 antibody or antigen-binding fragment thereof includes a light chain variable domain (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10- VL-CDR3 (SEQ ID NO: 11), a heavy chain variable domain (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDR1 D31N (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, relative to the CDR sequences of 3E10-D31N variant (SEQ ID NOs: 15- 18 and 22-24), selected from, but not limited to, a G to S substitution at position 5 of VH CDR2, a T to S substitution at position 14 of VH CDR2, an S to T substitution at position 5 of VL CDR1, an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2.
  • CDRs complementarity determining regions
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.1 (SEQ ID NO: 26) or 3E10-VH-CDR2.2 (SEQ ID NO: 27).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l l), and VH CDRs 1 and 3 (SEQ ID NOs:3 and 5) according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ ID NOs: 15 and 18) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.1 (SEQ ID NO: 28) or 3E10-VL-CDR1.2 (SEQ ID NO: 29).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs: 10 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CRDs 1 -3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10- D31N variant.
  • an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.1 (SEQ ID NO: 30).
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 11), and VH CDRs 1-3 (SEQ ID NOs: 3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:22 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18)according to the 3E10-D31N variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.3 (SEQ ID NO: 31).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 1 and 3 (SEQ ID NOs:3 and 5) according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragmentthereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ ID NOs: 15 and 18) according to the 3E10- D31N.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.3 (SEQ ID NO: 32).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs: 10 and 11), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10- D3 IN variant.
  • an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof, includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.2 (SEQ ID NO: 33).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:22 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10- D31N variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDR1.C1 (SEQ ID NO: 34), 3E10-VH-CDRl.c2 (SEQ ID NO: 35), 3E10-VH-CDRl.c3 (SEQ ID NO: 36), 3E10-VH-CDRl.c4 (SEQ ID NO: 37), or 3E10-VH- CDRl.c5 (SEQ ID NO: 38).
  • VH CDR1 comprising the amino acid sequence of 3E10-VH-CDR1.C1 (SEQ ID NO: 34), 3E10-VH-CDRl.c2 (SEQ ID NO: 35), 3E10-VH-CDRl.c3 (SEQ ID NO: 36), 3E10-VH-CDRl.c4 (SEQ ID NO: 37), or 3E10-VH- CDRl.c5
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 2 and 3 (SEQ ID NOs:4 and 5) according to the parent 3E10 antibody.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.cl (SEQ ID NO: 39), 3E10-VH-CDR2.c2 (SEQ ID NO: 40), or 3E10-VH-CDR2.c3 (SEQ ID NO: 41).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 -3 (SEQ ID NOs:9-l l), and VH CDRs 1 and 3 (SEQ ID NOs:3 and 5) according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ IDNOs: 15 and 18) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3.cl (SEQ ID NO: 42), 3E10-VH-CDR3.c2 (SEQ ID NO: 43), or 3E10-VH-CDR3.c3 (SEQ ID NO: 44).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l l), and VH CDRs 1 and 2 (SEQ ID NOs:3 and 4) according to the parent 3E10 antibody.
  • the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 2 (SEQ ID NOs: 15 and 17) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRl.cl (SEQ ID NO: 45), 3E10-VL-CDRl.c2 (SEQ ID NO: 46), 3E10-VL-CDR1.C3 (SEQ ID NO: 47), 3E10-VL-CDRl.c4 (SEQ ID NO: 48), 3E10-VL- CDRl.c5 (SEQ ID NO: 49), or 3E10-VL-CDRl.c6 (SEQ ID NO: 50).
  • VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRl.cl (SEQ ID NO: 45), 3E10-VL-CDRl.c2 (SEQ ID NO: 46), 3E10-VL-CDR1.C3 (SEQ ID NO: 47), 3E10-VL-CDRl.c4 (
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:10 and 1 1), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CRDs 1-3 (SEQ ID NOs: 15, 17, 18) according to the 3E10-D31N variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.cl (SEQ ID NO: 51).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:22 and 24), and VH CDRs 1-3 (SEQ ID NOs:15, 17 and 18) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3.cl (SEQ ID NO: 52), 3E10-VL-CDR3.c2 (SEQ ID NO: 53), 3E10-VL-CDR3.c3 (SEQ ID NO: 54), 3E10-VL-CDR3.c4 (SEQ ID NO: 55), 3E10-VL- CDR3.c5 (SEQ ID NO: 56), or 3E10-VL-CDR3.c6 (SEQ ID NO: 57).
  • VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3.cl (SEQ ID NO: 52), 3E10-VL-CDR3.c2 (SEQ ID NO: 53), 3E10-VL-CDR3.c3 (SEQ ID NO: 54), 3E10-VL-CDR3.c4 (SEQ ID NO: 55),
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:9 and 10), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:22 and 23), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10-D31N variant.
  • an antibody-payload conjugate comprising a humanized
  • 3E10 antibody or antigen-binding fragment thereof includes no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 CDR amino acid substitutions of the CDR amino acid substitutions described above. Further examples of 3E10 variant CDR sequences are described herein (SEQ ID NOs:58-63).
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO: 58).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 2 and 3 (SEQ ID NOs:4 and 5) according to the parent 3E10 antibody.
  • an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO: 59).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 1 and 3 (SEQ ID NOs: 3 and 5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ ID NOs: 15 and 18) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO: 60).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 1 and 2 (SEQ ID NOs:3 and 4) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 2 (SEQ ID NOs: 15 and 17) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO: 61).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs: 10 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CDRs 1-3 (SEQ ID NOs:15,17, and 18) according to the 3E10-D31N variant.
  • an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO: 62).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 1 1), and VH CDRs 1 -3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs: 22 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO: 63).
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:9 and 10), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody.
  • the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:9 and 10), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10-D3 IN variant.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of 3E10-VL-hl (SEQ ID NO:85), 3E10- VL-h2 (SEQ ID NO:86), 3E10-VL-h3 (SEQ ID NO:87), 3E10-VL-h4 (SEQ ID NO:88), 3E10- VL-h5 (SEQ ID NO:89), and 3E10-VL-h6 (SEQ ID NO: 90), where the light chain variable domain (3E10-VL) further comprises one or more amino acid residues selected from proline (Pro) at position 15, threonine (Thr) at position 22, tyrosine (Tyr) at position 49, Thr at position 74, asparagine (Asn) at position 76
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 5 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NOTO), 3E10-VL-CDR3 (SEQ ID NO: 11).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 4 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NOTO), 3E10-VL-CDR3 (SEQ ID NOT 1).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 3 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 2 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 1 amino acid substitution relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a lysine (Lys) residue at position 49 of the 3E10-VL according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a glutamic acid (Glu) residue at position 81 of the 3E10-VL according to Kabat numbering.
  • Glu glutamic acid
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a proline (Pro) residue at position 15 of the 3E10-VL according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a valine (Vai) residue at position 104, of the 3E10-VL according to Kabat numbering.
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of 3E10-VH-hl (SEQ ID NO:64), 3E10- VH-h2 (SEQ ID NO:65), 3E10-VH-h3 (SEQ ID NO:66), 3E10-VH-h4 (SEQ ID NO:67), 3E10- VH-h5 (SEQ ID NO:68), 3E10-VH-h6 (SEQ ID NO:69), and 3E10-VH-h7 (SEQ ID NO:70), where the heavy chain variable domain (3E10-VH) further comprises one or more amino acid residues selected from glutamine (Gin) at position 13, leucine (Leu) at position 18, arginine (Arg) at position 19, glycine (Glycine (Glycta
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 5 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO 5).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 4 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO:5).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 3 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO:5).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes set of 3E10-VH CDRs comprising no more than 2 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH- CDR3 (SEQ ID NO:5).
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 1 amino acid substitution relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH- CDR3 (SEQ ID NO 5)
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 5, 4, 3, 2, or 1 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
  • the present disclosure provides an antibody -payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with an arginine (Arg) residue at position 18 of the 3E10-VH according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a (Lys) residue at position 19 of the 3E10-VH according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with an alanine (Ala) residue at position 49 of the 3E10-VH according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a glutamine (Gin) residue at position 13, of the 3E10-VH according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a leucine (Leu) residue at position 108, of the 3E10-VH according to the Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a valine (Vai) residue at position 109, of the 3E10-VH according to Kabat numbering.
  • the present disclosure provides an antibody -payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a serine (Ser) residue at position 113, of the 3E10-VH according to Kabat numbering.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof with a fragment crystallizable (Fc) region.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof with an Fc region selected from a human IgGl Fc, a human IgG2a Fc, a human IgG2b Fc, a human IgG3 Fc, and a human IgG4 Fc.
  • the present disclosure provides an antibody-payload conjugate comprising humanized 3E10 antibodies or variants thereof, or antigen-binding fragments thereof comprising a heavy chain constant domain (CH).
  • CH heavy chain constant domain
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof comprises an Fc region selected from a human yl CHI, a human ⁇ 2 CHI, a human ⁇ 3 CHI, and a human ⁇ 4 CHI.
  • the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain constant domain (CL).
  • a humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain constant domain (CL).
  • an antibody-payload conjugate comprising a humanized 3E10 antibody or variant comprising an Fc region selected from the group consisting of a human L CL and a human K CL.
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof comprising a combination of a light chain variable domain (VL) and a heavy chain variable domain (VH) selected from 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-hl (SEQ ID NO:64), 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-h2 (SEQ ID NO:65), 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-h3 (SEQ ID NO:66), 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-h4 (SEQ ID NO:
  • an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof comprises a combination of a light chain variable domain (VL) of 3E10-VL-h6 (SEQ ID NO:90) and a heavy chain variable domain (VH) of 3E10- VH-h6 (SEQ ID NO:69).
  • VL light chain variable domain
  • VH heavy chain variable domain
  • Antibodies useful in the compositions, conjugates, and methods described herein include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody.
  • the variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR).
  • CDRs complementarity determining regions
  • FR framework
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. Therefore, the antibodies typically contain at least the CDRs necessary to maintain DNA binding.
  • the 3E10 antibody is typically a monoclonal 3E10, or a variant, derivative, fragment, fusion, or humanized form thereof that binds the same or different epitope(s) as 3E10.
  • a deposit according to the terms of the Budapest Treaty of a hybridoma cell line producing monoclonal antibody 3E10 was received on September 6, 2000, and accepted by, American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209, USA, and given Patent Deposit Number PTA-2439.
  • ATCC American Type Culture Collection
  • the antibody can have the same or different epitope specificity as monoclonal antibody 3E10 produced by ATCC No. PTA 2439 hybridoma.
  • the antibody can have the paratope of monoclonal antibody 3E10.
  • the antibody can be a single chain variable fragment of 3E10, or a variant, e.g., a conservative variant thereof.
  • the antibody can be a single chain variable fragment of 3E10 (3E10 Fv), or a variant thereof.
  • the heavy chain complementarity determining regions can be defined according to the IMGT system.
  • the complementarity determining regions (CDRs) as identified by the IMGT system include CDR Hl.3 (original sequence): GFTFSDYG (SEQ ID NO:989); CDR Hl.4 (with D31N mutation): GFTFSNYG (SEQ ID NO:990); CDR H2.2: ISSGSSTI (SEQ ID NO:991) and variant ISSSSSTI (SEQ ID NO:992); CDR H3.2: ARRGLLLDY (SEQ ID NO:993).
  • the light chain complementarity determining regions can be defined according to the IMGT system.
  • the complementarity determining regions (CDRs) as identified by the IMGT system include CDR LI.2 KSVSTSSYSY (SEQ ID NO:994) and variant KTVSTSSYSY (SEQ IDNO:995); CDRL2.2: YAS; CDRL3.2: QHSREFPWT (SEQ ID NO: 996).
  • compositions, conjugates, and methods typically utilize antibodies that maintain the ability to penetrate cells, and optionally nuclei.
  • the mechanisms of cellular internalization by autoantibodies are diverse. Some are taken into cells through electrostatic interactions or FcR-mediated endocytosis, while others utilize mechanisms based on association with cell surface myosin or calreticulin, followed by endocytosis (Ying-Chyi et al., Eur J Immunol 38, 3178-3190 (2008), Yanase et al., J Clin Invest 100, 25-31 (1997)).
  • the 3E10 antibodies and antigen-binding fragments thereof can transit cellular membranes via an equilibrative nucleoside (ENT) transporter.
  • 3E10 transits cellular membranes via an ENT1, ENT2, ENT3 or ENT4 transporter (See, e.g., WO 2015/106290 Al and WO 2016/033324 Al, each of which is incorporated by reference herein, in its entirety).
  • 3E10 penetrates cells in an Fc- independent mechanism (as evidenced by the ability of 3E10 fragments lacking an Fc to penetrate cells) but involves presence of the nucleoside transporter ENT2 (Weisbart et al., Sci Rep 5: 12022. doi: 10.1038/srepl2022.
  • the antibodies utilized in the disclosed compositions, conjugates, and methods are ones that penetrates cells in an Fc- independent mechanism and involve the presence of the nucleoside transporter ENT2.
  • variants 10 and 13 penetrated nuclei very well compared to the murine antibody.
  • NLS nuclear localization signals
  • RASKTVSTSSYSYMHWYQQKPGQPPKLLIKY (SEQ ID NO: 139); or RVTITCRASKSVSTSSYSYMHWYQQKPGKAPKL (SEQ ID NO: 140).
  • the disclosed antibodies may include the sequence of any one of SEQ ID NOs: 138-142, or fragments and variants thereof (e.g., at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity with any one of SEQ ID NOs: 138-142) that can translocate into the nucleus of a cell.
  • Presence of an NLS indicates that a humanized 3E10 antibody or antigen binding fragment thereof may cross the nuclear envelope via the nuclear import pathway.
  • the NLS improves importation by interacting with one or more members of the import pathway.
  • the NLS can bind to importin- , an importin- ⁇ /importin- ⁇ heterodimer, or a combination thereof.
  • compositions and methods utilize humanized 3E10 antibodies and ENT2-binding fragments thereof that maintain the ability to bind nucleic acids such as DNA, RNA.
  • the disclosed humanized 3E10 antibodies include some or all of the underlined NAB1 sequences.
  • the humanized 3E10 antibodies include a variant sequence that has an altered ability of bind nucleic acids.
  • the mutations e.g., substitutions, insertions, and/or deletions
  • the mutations improve binding of the antibody to nucleic acids such as DNA, RNA, or a combination thereof.
  • the mutations are conservative substitutions.
  • the mutations increase the cationic charge of the NAB1 pocket.
  • Additional example variants include mutation of aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R), which modeling indicates expands cationic charge, or lysine (3E10-D3 IK) which modeling indicates changes charge orientation.
  • the 3E10 binding protein includes a D31R or D3 IK substitution.
  • Additional example variants include mutation of arginine (R) 96 to asparagine (N), and/or serine (S) 30 to aspartic acid (D) alone or in combination with D3 IN, D31R, or D3 IK.
  • FIG. 1 IB is an illustration showing molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues illustrated with punctate dots.
  • substitutions can be included in any combination.
  • the sequence having two or three substitutions at any combination of residues 31, 30, and 96 are expressly provided.
  • the sequence has 3 IN, 3 IK, or 31R alone or in combination with 30D, and without the R96N substitution.
  • the residue corresponding to 96 is not N, and in more specific embodiments remains R.
  • the antibody-oligonucleotide conjugates (AOCs) provided herein comprises a linker.
  • the linker (L) described herein can be used to link or conjugate the 3E10 antibody or antigen-binding fragment thereof to an oligonucleotide, e.g., an siRNA or antisense oligonucleotide.
  • the term “linker” as used herein includes, without limitation, any known linker for use in antibody-oligonucleotide-conjugates known in the art.
  • the AOC comprises, one, two, three, four, or more linkers.
  • one or more amino acids suitable conjugation of a 3E10 antibody or antigen-binding fragment thereof provided herein are selected from lysine, cysteine, histidine, arginine, aspartic acid, glutamine, serine, threonine and tyrosine.
  • one or more amino acids suitable for conjugation are introduced by substitution of one or more amino acids in the 3E10 antibody or antigen-binding fragment thereof.
  • the one or more conjugated amino acids are lysine or arginine, and conjugation is conducted via amine conjugation.
  • one or more conjugated amino acids are glutamine (Gin) and conjugation is conducted via transglutaminase (TGase) mediated enzymatic conjugation.
  • one or more conjugated amino acids are cysteine (Cys) and conjugation is conducted via thiol conjugation.
  • NHS-PEG reagent can be used to modify primary amines in a 3E10 antibody or antigen-binding fragment thereof provided herein.
  • a composition comprising an AOC as described herein has an average DAR of at least 4 (an average of at least four drug moieties attached to each antibody). In some embodiments, such a composition has an average DAR of at least 6. In some embodiments, such a composition has an average DAR of at least 8. In some embodiments, such a composition has an average DAR of at least 10. In some embodiments, such a composition has an average DAR of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more. [0429] In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 12.
  • a composition comprising an AOC as described herein has an average DAR of no more than 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 4.
  • a composition comprising an AOC as described herein has an average DAR of from 2 to 4. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 4.
  • a composition comprising an AOC as described herein has an average DAR of from 3 to 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 10.
  • a composition comprising an AOC as described herein has an average DAR of from 4 to 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 6 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 6 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 6 to 12.
  • a method of site-specific conjugation is by means of transglutaminase.
  • Transglutaminases which also include bacterial transglutaminase (BTG) are a family of enzymes which catalyse the formation of a covalent bond between the y-carbonyl-amide group of glutamines and the primary amine group of lysines.
  • a peptide or antibody can be a substrate for transglutaminase according to the methods of the present disclosure.
  • the peptide or antibody contains a Gin or a Lys residue, and in particular a Gin residue.
  • the peptide or antibody is not a transglutaminase substrate, so one or more Gin or Lys residues, and in particular Gin residues, are inserted into the peptide or antibody sequence to make the peptide a substrate for transglutaminase.
  • a Gin or Lys residue may be inserted at any position in the peptide or antibody sequence, however, it is preferably inserted at a position where the physiological properties, such as the therapeutic activity of the peptide is not affected to a degree where the peptide is not useful anymore, e.g., in a therapeutic intervention. Insertions of amino acid residues in peptides can be brought about by standard techniques known to persons skilled in the art, such as post-translational chemical modification or transgenic techniques, as described, for example, in US Patent No. 11,123,439 and US 2016/0355859, the contents of which are hereby incorporated by reference.
  • transglutaminases also accept substrates other than lysine as amine donor, they are used to modify proteins including antibodies at suitable acceptor glutamines (Josten et al., J. Immunol. Methods 240, 47-54 (2000); Mindt et al., Bioconjugate Chem. 19, 271- 278 (2008); Dennler et al., in Antibody Drug Conjugates (Ducry, L , Ed.), pp 205-215, Humana Press. (2013), the contents of which are incorporated herein by reference).
  • Transglutaminases have been used for the conjugation of drugs to antibodies containing artificial glutamine tags which are acceptor glutamine residues which have been introduced into the antibody by genetic engineering (Strop et al., Chem. Biol. 20, 161-167 (2013)). Furthermore, the conserved glutamine residue Q295 (Kabat EU numbering) of the constant region of the heavy chain of antibodies is the only y-carbonyl-amide donor for the bacterial transglutaminase (EC 2.3.2.13) in the backbone of aglycosylated IgGl molecules, and is thus an acceptor glutamine, whereas no acceptor glutamine is present in the backbone of IgGl when the antibody has been glycosylated at position N297 (Kabat EU numbering) of the heavy chain.
  • bacterial transglutaminase can be used for the conjugation of an amine-donor substrate, for example a drug-linker construct, at an acceptor glutamine residue of an antibody.
  • acceptor glutamines can be introduced by engineering of the antibody by mutations or by the generation of aglycosylated antibodies.
  • aglycosylated antibodies can be introduced by deglycosylation using N-glycosidase F (PNGase F) or by mutation of N297 of the glycosylation site of the heavy chain (Kabat EU numbering) to any other amino acid except N.
  • PNGase F N-glycosidase F
  • Kabat EU numbering mutation of N297 of the glycosylation site of the heavy chain
  • the enzymatic conjugation of such aglycosylated antibodies using bacterial transglutaminase has been described for aglycosylated antibody variants containing the mutations N297D, N297Q or N297S (see U.S. Pat Nos. US 9,764,038 and US 9,764,038, the contents of which are incorporated by reference).
  • the enzymatic conjugation of such aglycosylated antibodies by means of transglutaminase generally affords AOCs having a drug (oligonucleotide) antibody ratio (DAR or OAR) of 2, in which both heavy chains are specifically functionalized at position Q295 (Kabat EU numbering). Only mutation N297Q of the heavy chain affords an additional conjugation site per heavy chain.
  • the conjugation of such variants leads to AOCs having a DAR of 4, in which both heavy chains are specifically functionalized at positions Q295 and Q297.
  • the chemical modification strategy utilized to create antibody- payload conjugates described herein is lysine conjugation.
  • Lysine residues in proteins, e.g., antibodies possess a primary amine group (-NH2) in their side chains, making them suitable targets for chemical modification.
  • This primary amine group can react with various chemical reagents, including small molecules or polymers (e.g., cleavable and non-cleavable linkers).
  • lysine conjugation can be either site-specific or random.
  • site-specific conjugation specific lysine residues within a protein, e.g., antibody, can be targeted ensuring precise control over the modification.
  • random conjugation involves modifying lysine residues without selectivity.
  • VL K53 is the most modified lysine.
  • the conjugation strategy is selected from one of the following:
  • the linker moiety is conjugated to the antibody or antigen binding fragment therein through an amine linkage at one or more surface-exposed lysine residues on the antibody or antigen binding fragment thereof.
  • lysine conjugation is a random process, with respect to which lysines are conjugated to the liker- payload.
  • some preference for conjugation at certain lysines can occur due to the context of the primary, secondary, ternary, and/or quaternary structure surrounding a particular lysine residue.
  • Many different chemistries are known in the art for attaching payloads to proteins at lysine groups.
  • activated esters on the drug-linker complexes can react with the antibody lysine residues and achieve conjugation via amide bonds, or stable amidine bonds can be generated on an antibody by the reaction of imido ester compounds, such as Traut’s reagent, with antibody lysine residues.
  • O-succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters
  • the present disclosure provides pharmaceutical compositions comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L- Pr) q , where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein, L is a linker as described herein, P is an oligonucleotide moiety as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, in which L is conjugated to A through lysine moieties.
  • AOC antibody-oligonucleotide conjugate
  • a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein will have an average DAR of at least 4 (an average of at least four drug moieties attached to each antibody).
  • such a composition will have an average DAR of at least 6.
  • such a composition will have an average DAR of at least 8.
  • such a composition will have an average DAR of at least 10.
  • such a composition will have an average DAR of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more.
  • a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 1 and q is at least 4. In some embodiments, r is 1 and q is at least 6. In some embodiments, r is 1 and q is at least 8. In some embodiments, r is 1 and q is at least 10. In some embodiments, r is 1 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more.
  • a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 2 (e.g., the linker is a branched linker) and q is at least 2. In some embodiments, r is 2 and q is at least 3. In some embodiments, r is 2 and q is at least 4. In some embodiments, r is 2 and q is at least 5. In some embodiments, r is 2 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more.
  • a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, the linker is highly branched, e.g., r is at least 3. In some embodiments, r is at least 4. In some embodiments, r is at least 5, 6, 7, 8, or more.
  • lysine conjugation can be either site-specific or random. In site- specific conjugation, specific lysine residues within a protein, e.g., antibody, can be targeted ensuring precise control over the modification. In contrast, random conjugation involves modifying lysine residues without selectivity.
  • Example 6 shows greater uptake of 3E10-D31N monoclonal antibody (V66) oligonucleotide conjugates utilizing transglutaminase-mediated enzymatic conjugation in A427 cells, and in Example 7, which utilize hindered disulfide linker (SPDMV) conjugate (transglutaminase- SPDMV), and imparted the highest exon-skipping (-15%), representing a greater than 10X improvement over lysine-PEG8 AOC.
  • SPDMV hindered disulfide linker
  • the linker moiety is conjugated to the antibody or antigen binding fragment therein through a sulfide linkage at one or more surface-exposed cysteine residues on the antibody or antigen binding fragment thereof. In some embodiments of AOCs described herein, the linker moiety is conjugated to the antibody or antigen binding fragment therein through a thiol side chain of one or more cysteine residues on the antibody or antigen binding fragment thereof.
  • antibodies do not possess free thiols, and all cysteine residues form disulfide bonds.
  • human IgGl which is commonly used in modem ADCs, there are 4 interchain and 12 intrachain disulfide bonds.
  • the 4 interchain disulfides which are generally not critical for structural stability of IgGl, can be selectively reduced under mild conditions to give 2, 4, 6, or 8 free thiols while keeping the 12 intrachain disulfides intact. Due to the limited number of conjugation sites and the distinct reactivity of the thiol group, cysteine-based conjugation allows for controlled DAR and heterogeneity. Engineered Cys residues can also be used for site specific conjugation without the partial reduction of the endogenous disulfide bonds using, e.g., EnCys-mAb technology. Many different chemistries are known in the art for attaching payloads to proteins at cystine groups.
  • 8 nucleophilic cysteine residues can first be liberated from the reduced inter-chain disulfide bonds via reducing agents and later conjugated with drug-linker complexes.
  • This approach generates ADCs with heterogeneous conjugation sites and a different number of drugs attached, resulting in a drug to antibody ratio (DAR) ranging from 0 ⁇ 8.
  • DAR drug to antibody ratio
  • DTT dithiothreitol
  • TCEP tri s(2-carboxy ethyl) phosphine
  • DTNB 5,5 ’ -dithiobis (2 -nitrobenzoic acid)
  • the present disclosure provides pharmaceutical compositions comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L- Pr) q , where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein, L is a linker as described herein, P is an oligonucleotide moiety as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, in which L is conjugated to A through cysteine moi eties.
  • AOC antibody-oligonucleotide conjugate
  • cysteine-based conjugation can be used to generate AOC molecules with a controlled DAR and heterogeneity.
  • a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein will have an average DAR of at least 2.
  • such a composition will have an average DAR of at least 3.
  • such a composition will have an average DAR of at least 4.
  • such a composition will have an average DAR of at least 5.
  • such a composition will have an average DAR of at least 6.
  • such a composition will have an average DAR of at least 7.
  • such a composition will have an average DAR of about 8.
  • such a composition will have an average DAR of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or about 8.
  • a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 1 and q is at least 2. In some embodiments, r is 1 and q is at least 3. In some embodiments, r is 1 and q is at least 4. In some embodiments, r is 1 and q is at least 5. In some embodiments, r is 1 and q is at least 6. In some embodiments, r is 1 and q is at least 7. In some embodiments, r is 1 and q is about 8. In some embodiments, r is 1 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8.
  • a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 2 (e.g., the linker is a branched linker) and q is at least 2. In some embodiments, r is 2 and q is at least 3. In some embodiments, r is 2 and q is at least 4. In some embodiments, r is 2 and q is at least 5. In some embodiments, r is 2 and q is at least 6. In some embodiments, r is 2 and q is at least 7. In some embodiments, r is 2 and q is about 8.
  • r is 2 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or about 8.
  • a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, the linker is highly branched, e.g., r is at least 3.
  • r is at least 4.
  • r is at least 5, 6, 7, 8, or more.
  • the linker moiety is conjugated to the antibody or antigen binding fragment therein through the primary amide side chain of one or more glutamine residues on the antibody or antigen binding fragment thereof.
  • transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR of 2 (one conjugation site per heavy chain)
  • ADCs with a defined DAR of 2 (one conjugation site per heavy chain)
  • the present disclosure provides pharmaceutical compositions comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L- P r) q , where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein, L is a linker as described herein, P is a payload moiety as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, in which L is conjugated to A through glutamine moieties.
  • AOC antibody-oligonucleotide conjugate
  • a composition comprising an AOC where glutamine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein will have an average Drug Antibody Ratio (DAR) of about 2.
  • DAR Drug Antibody Ratio
  • such a composition will have an average DAR of at least 2.
  • such a composition will have an average DAR of at least 3.
  • such a composition will have an average DAR of about 4.
  • such a composition will have an average DAR of about 2, at least 2, at least 3, or about 4.
  • a composition comprising an AOC where glutamine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein r is 1 and q is about 2. In some embodiments, r is 1 and q is at least 2. In some embodiments, r is 1 and q is at least 3. In some embodiments, r is 1 and q is about 4. In some embodiments, r is 1 and q is about 2, at least 2, at least 3, or about 4. In some embodiments, a composition comprising an AOC where glutamine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 2 (e.g., the linker is a branched linker) and q is about 2.
  • r is 2 and q is at least 2. In some embodiments, r is 2 and q is at least 3. In some embodiments, r is 2 and q is about 4. In some embodiments, r is 2 and q is about 2, at least 2, at least 3, or about 4. In yet other embodiments, a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, the linker is highly branched, e.g., r is at least 3. In some embodiments, r is at least 4. In some embodiments, r is at least 5, 6, 7, 8, or more.
  • the linker is a cleavable linker.
  • cleavable linker refers to a linker which can connect two or more molecules and then be cleaved once exposed to an agent.
  • Cleavable linkers can include chemically or enzymatically unstable or degradable linkages.
  • Cleavable linkers generally rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell.
  • Cleavable linkers generally incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker is non-cleavable.
  • the cleavable linker is an acid-labile linker, a phosphatase linker, glucuronidase linker, a cathepsin-B cleavable linker, a cathepsin-L cleavable linker, a protease-sensitive linker, a photo-labile linker, or a disulfide (SPDMV) transglutaminase- containing linker.
  • SPDMV disulfide
  • the cleavable linker is selected from the group consisting of succinyl, O-succinyl, 4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)toluene, sulfosuccinimidyl 6-(3’-(2-pyridyldithio)propionamido)hexanoate, N-succinimidyl-3-(-2-pyridyldithio)- proprionate, succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate, 3-(2-pyridyldithio)- propionyl hydrazide, S-(2-thiopyridyl)-L-cysteine, N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (CAS: 115088-06-7), N-
  • the cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain.
  • the linker length of the polyethylene glycol (PEG) chain is 4 PEG molecules, i.e., PEG4.
  • the PEG chain is PEG8.
  • the PEG chain is PEG12.
  • the cleavable homo-bi-functional linker is selected from the group consisting of DSP (Lomant’s Reagent) (CAS: 57757-57-0) and Acid-PEG4-S-S-PEG4- Acid (CAS: 2055015-40-0).
  • linkers which contain cleavable disulfide bonds include, but are not limited to “DPDPB”, l,4-di-[3’-(2’-pyridyldithio) propionamido]butane; “SADP”, (N- succinimidyl (4-azidophenyl) l,3’-dithio propionate); “Sulfo-SADP” (Sulfosuccinimidyl (4-azi- dophenyldithio) propionate; “DSP”-Dithio bis (succinimidylproprionate); “DTSSP”-3,3’-Dithio bis (sulfosuccinimidylpropionate); “DTBP” -dimethyl 3,3dithiobispropionimidate-2HCI.
  • linkers cleavable by oxidation include “DST’-disuccinimidyl tartarate; and “S-di-[3
  • the linker L is selected from:
  • the linker moiety is conjugated to the antibody or antigen binding fragment therein through a cathepsin-cleavable linker.
  • AOCs can enter cells via receptor-mediated endocytosis, during intracellular transit or trafficking, and thus they may ultimately encounter the acidic degradative environment of the lysosome which contains multiple proteases and other catalytic enzymes for breakdown of internalized biologic substances.
  • cleavable linkers therefore leverage an attribute of the lysosome for payload release such as lability to low pH environment, disulfide reducing environment, or cleavage by a lysosomal protease such as cathepsin B.
  • Protease cleavable linkers or (peptide linkers) typically contain a dipeptide sequence based on deduced canonical cleavage specificity for a given protease.
  • 3E10 and its derivatives are anti-DNA antibodies with the unique property of direct cell entry and subsequent trafficking to the nucleus of cells. This occurs via interaction of antibody:DNA complexes with the cell surface transporter ENT2 (equilibrative nucleoside transporter-2) and so does not require the classical endocytic mechanism employing clathrin or dynamin mediated internalization. Further, upon internalization, the antibody does not encounter early or late endosomes and, importantly, does not encounter the harsh environment of the lysosome. As a result, trafficking occurs directly to the nucleus. As such, 3E10 and its derivatives can serve as delivery vehicles for therapeutic payloads that are mechanistically active within the nucleus of target cells.
  • an AOC described herein comprises a cleavable linker for which the catalytic agent is present within the within the nucleus for selective release within that organelle.
  • cysteine protease Cathepsin L can also be present within the nucleus of cells. See, e.g., Goulet et al., “Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation,” Mol Cancer Res. 2007 Sep;5(9):899-907, incorporated herein by reference in its entirety.
  • This ubiquitous protease has previously been shown to be present primarily in lysosomes as well as in secreted form.
  • Translation initiation within the cathepsin L mRNA for both mouse and human cells has been shown to take place at alternative internal start sites resulting in a polypeptide lacking an NH2 -terminal signal peptide.
  • Other cathepsins typically identified in lysosomes have also been identified in the nucleus, including cathepsin D, cathepsin V, and cathepsin B.
  • a nuclear form of the cysteine protease cathepsin-L is found in mouse tumor cells (and also in human tumor cells) (Goulet et al., “Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation,” Mol Cancer Res. 2007 Sep;5(9):899-907; and Soond et al., “Lost or Forgotten: The nuclear cathepsin protein isoforms in cancer,” Cancer Lett. 2019 Oct 10;462:43-50, each incorporated herein by reference in their entireties. Indeed there have been identified several proteases identified that localize to the nucleus but cathepsin-L is most prominent. Further, in cancer cells cathepsin-L appears to play a significant role in cell cycle progression and importantly tumor progression and metastasis.
  • AOCs disclosed herein comprise dipeptides that are substrates for proteolytic cleavage by nuclear localized cathepsins, optionally cathepsin L, cathepsin S, cathepsin D, cathepsin V, and/or cathepsin B.
  • the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-P r ) q , where: A is a 3E 10 antibody or antigen-binding fragment thereof as described herein (e.g., as described in the section titled 3E10 Antibodies and Antigen-Binding Fragments), L is a cathepsin-cleavable linker, P is a payload moiety, r is an integer from 1 to 4 and q is an integer from 1 to 16. In some embodiments, L is a cathepsin L- cleavable linker. In some embodiments, L is a cathepsin S-cleavable linker.
  • AOC antibody-oligonucleotide conjugate
  • L is a cathepsin D-cleavable linker. In some embodiments, L is a cathepsin B-cleavable linker. In some embodiments, L is a cathepsin V-cleavable linker.
  • L comprises a dipeptide selected from Phe-Gln, Val-Gln, Leu- Gin, Tyr-Met, Phe-Arg, Phe-Gly, Trp-Thr, Tyr-Gly, Phe-Thr, Val-Gly, Val-Cit, Val-Arg, Thr-Thr, and Val-Thr.
  • L further comprises one or more PEG molecules.
  • the linker is a non-cleavable linker.
  • “non- cleavable linker” refers to linkers where the release of the biologically active molecule does not depend on, for example, the differential properties between the plasma and some cytoplasmic compartments, or whether the linker has a physical property that permits enzymatic cleavage or chemical cleavage.
  • Non-cleavable linkers can be alkylene chains, or can be polymeric in nature, such as, for example, those based upon polyalkylene glycol polymers, amide polymers, or can include segments of alkylene chains, polyalkylene glycols and/or amide polymers.
  • the non-cleavable linker is selected from DBCO-C6-NHS ester (CAS: 1384870-47-6) and DBCO-PEG8-NHS ester.
  • the non-cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain.
  • the non- cleavable homo-bi-functional linker is selected from DSS (CAS: 68528-80-3) and Bis-PEG8-NHS ester.
  • non-cleavable linkers can include N-succinimidyl (4-iodoacetyl)- aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate, dichlorotriazinic acid, N- succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide), N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), or N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC).
  • NHS-PEG4-maleimide N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate
  • sulfoSMCC N-sulfosuccinimid
  • Non-cleavable linkers are “Sulfo-LC-SMPT”- (sulfosuccinimidyl 6-[alphamethyl-alpha-(2-pyridylthio) toluamido ⁇ hexanoate; “SMPT” - 4- succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)toluene; “ABH”-Azidobenzoyl hydrazide; “NHS-ASA”-N-Hydroxysuccinimidyl-4-azi dosalicyclic acid; “SASD”- Sulfosuccinimidyl 2-(pazidosali- cylamido)ethyl-l,3-dithiopropionate; “APDP”-N- ⁇ 4-(p-azi dosalicylamido) buthy ⁇ -3’ (2’-pyidyldithio)propion
  • Bifunctional chemical linkers include 4-succinimidyl-oxycarbonyl-C-(2- pyridyldithio) toluene; sulfosuccinimidyl-6-O-methyl-O-(pyridyldithiol)-toluamidohe-xanoate; N-succinimidyl-3(2-pyridyldithio)-propri onate; succinimidyl -6-3(-(-2-pyridyldithio)- proprionamidohexanoate; sulfosuccinimidyl-6-3 (-(-2-pyridyldithio)-propionamido hexanoate; 3- (2-pyridyldithio)-propionyl hydrazide, Ellman’s reagent, dichlorotriazinic acid, S-(2-thiopyridyl)- Lcysteine
  • cross-linking agents there are a large number of chemical cross-linking agents that are known to those skilled in the art and useful for cross-linking portions of a conjugate.
  • the cross-linking agents are heterobifunctional cross-linkers, which can be used to link molecules in a stepwise manner.
  • Heterobifunctional crosslinkers provide the ability to design more specific coupling methods for conjugating, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers.
  • a wide variety of heterobifunctional cross-linkers are known in the art, including succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC), m-Maleimidobenzoyl N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB); succinimidyl 4-(p-ma-30 leimidophenyl) butyrate (SMPB); l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)- tolune (SMPT); N-succinimidyl 3-(2-pyridyldithio) propi onate (SPDP); succinimidyl 6-[3-(2- pyridyl 4-(
  • cross-linking agents having N- hydroxysuccinimide moieties can be obtained as the N-hydroxysulfosuccinimide analogs, which generally have greater water solubility.
  • those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo.
  • heterobifunctional cross-linkers there exists a number of other cross-linking agents including homobifunctional and photoreactive cross- linkers.
  • DSS Disuccinimidyl subcrate
  • BMH bismaleimidohexane
  • DMP dimethylpimelimidate 2 HC1
  • BASED bis-[B-(4- azidosalicylamido)ethyl]disul fide
  • SANPAH N-succinimidyl-6(4’-azido-2’-nitrophe nylamino)hexanoate
  • One class of heterobifunctional cross-linkers contain the primary amine reactive group, N-hydroxysuccinimide (NHS), or its water soluble analog N- hydroxysulfosuccinimide (sulfa-NHS).
  • NHS N-hydroxysuccinimide
  • sulfa-NHS water soluble analog N- hydroxysulfosuccinimide
  • Primary amines lysine epsilon groups
  • alkaline pH is unprotonated and react by nucleophilic attack on NHS or sulfa-NHS esters. This reaction results in the formation of an amide bond, and release of NHS or sulfa-NHS as a byproduct.
  • Another reactive group useful as part of a hetero bifunctional cross-linker is a thiol reactive group.
  • Common thiol reactive groups include maleimides, halogens, and pyridyl disulfides.
  • Maleimides react specifically with free sulfhydryls (cysteine residues) in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions.
  • Halogens iodo acetyl functions
  • the universal antibody-oligonucleotide conjugate (AOC) provided herein comprises a linker attachment schema selected from the group consisting of: conjugation to exposed lysines, cysteine conjugation following modest reduction of 3E10, engineering free cysteines (e.g., at the C -terminus), site-specific transglutaminase linkage, wherein the linkage can further comprise click chemistry, and any combination thereof.
  • an AOC provided herein further comprises a linker attachment schema comprising engineering free cysteines (e.g., at the C -terminus).
  • an AOC provided herein comprises an antibody that is cysteine-engineered at the site of linker attachment.
  • an AOC provided herein comprises a cleavable linker comprising a cleavable disulfide bond.
  • an AOC provided herein comprises a linker wherein the linker is a hindered linker, wherein the linker comprises a carbon atom bearing a sulfur capable of forming a disulfide bond, and wherein the carbon atom is substituted with at least one substituent other than H.
  • the substituent comprises a hydrocarbyl or a substituted hydrocarbyl moiety.
  • the linker L is selected from:
  • the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-P r )q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is an ASO described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16.
  • AOC antibody-oligonucleotide conjugate
  • the term “therapeutic oligonucleotide” refers to an oligonucleotide that has a biological, a cytotoxic, or a therapeutic effect in a cell.
  • the therapeutic oligonucleotide is a functional nucleic acid, such as an oligonucleotide or a polynucleotide.
  • P is an ASO that mediates exon skipping.
  • an ASO that mediates exon skipping is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site of a pre-mRNA transcript.
  • an ASO that mediates exon skipping is a single stranded oligonucleotide capable of hybridizing to a donor splice site of a pre- mRNA transcript.
  • an ASO that mediates exon skipping is a single stranded oligonucleotide capable of hybridizing to an exonic splice enhancer element of a pre-mRNA transcript.
  • an ASO that mediates exon skipping induces exon skipping in the pre- mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • a nucleic acid sequence whose function is to be modulated is first identified. This may be, for example, a gene (or mRNA transcribed form the gene) whose expression is associated with a particular disorder or disease state, e.g., Duchenne muscular dystrophy.
  • target site(s) are those involved in mRNA splicing (i.e., splice donor sites, splice acceptor sites, or exonic splicing enhancer elements).
  • splicing branch points and exon recognition sequences or splice enhancers are potential target sites for modulation of mRNA splicing.
  • antisense oligonucleotides capable of binding to a selected target in the pre-mRNA to induce efficient and consistent exon skipping.
  • the antisense oligonucleotides and pre-mRNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • the term “complementary” is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.
  • sequence of an antisense molecule need not be 100% complementary to that of its target sequence to interfere with the normal function of the target DNA or RNA as well as to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment.
  • the length of an antisense oligonucleotide may vary so long as it is capable of binding selectively to the intended location within the pre-mRNA molecule.
  • the length of such sequences can be determined in accordance with selection procedures described herein.
  • the antisense oligomer will be from about 10 nucleotides in length up to about 50 nucleotides in length. It will be appreciated however that any length of nucleotides within this range may be used in the method.
  • the length of the antisense molecule is between 17 to 30 nucleotides in length.
  • the antisense oligomers used in the method may be adapted to minimize or prevent cleavage by endogenous RNase H. This property is highly preferred as the treatment of the RNA with the unmethylated oligonucleotides either intracellularly or in crude extracts that contain RNase H leads to degradation of the pre-mRNA: antisense oligomer duplexes. Any form of modified antisense molecules that is capable of bypassing or not inducing such degradation may be used in the present method.
  • An example of antisense oligomer which when duplexed with RNA are not cleaved by cellular RNase H is 2'-O-methyl derivatives. 2'-O-methyl-oligoribonucleotides are very stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo-counterparts.
  • Antisense oligonucleotides that do not activate RNase H can be made in accordance with known techniques, see, e.g., U.S. Pat. No. 5, 149,797. Such antisense oligonucleotides, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification which sterically hinders or prevents binding of RNase H to a duplex molecule containing the oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation.
  • antisense molecules that do not activate RNase H are available.
  • such antisense oligonucleotides wherein at least one, or all, of the inter-nucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates.
  • every other one of the internucleotide bridging phosphate residues may be modified as described.
  • such antisense oligonucleotides are oligonucleotides wherein at least one, or all, of the nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2- propenyl, and isopropyl).
  • a 2' lower alkyl moiety e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2- propenyl, and isopropyl.
  • every other one of the nucleotides may be modified as described.
  • oligonucleotide mimetics include, but not limited to, oligonucleotide mimetics.
  • oligonucleotides containing modified backbones or non-natural inter-nucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their inter-nucleoside backbone can also be considered to be oligonucleosides.
  • the oligonucleotide mimetics both the sugar and the inter- nucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid PNA
  • PNAs Peptide nucleic acids
  • the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached.
  • PNAs containing natural pyrimidine and purine bases hybridize to complementary oligomers obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993).
  • the backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications.
  • the backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.
  • Modified oligonucleotides may also contain one or more substituted sugar moi eties.
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • Another modification of the oligonucleotides of the disclosure involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di- hexadecyl-rac -glycerol or tri ethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thi
  • Chimeric antisense compounds or “chimeras,” in the context of this invention are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid. /.
  • the exon-skipping inducing oligonucleotides have a sequence selected from SEQ ID NO:99-103, as detailed in Figure 15, or any variation thereof disclosed herein.
  • the exon-skipping inducing oligonucleotides can be selected from the group consisting of SEQ ID NO: 150-398, as detailed in Table 2.
  • the exon-skipping inducing oligonucleotides can be selected from the group consisting of SEQ ID NO:410-988, as detailed in Table 3. It should be understood that the oligonucleotides disclosed in Table 2 and Table 3 are exemplary in nature, and in no way limiting the present invention.
  • Table 1 Example genes targeted by exon skipping oligos.
  • Table 2 Example sequences of oligos for use in treating various disorders.
  • the present disclosure provides an antibody-payload conjugate having the formula A-(L-P r )q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16.
  • L is a cathepsin-L cleavable linker
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60.
  • the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cathepsin-L cleavable linker is an un-branched linker.
  • the cathepsin-L cleavable linker is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms.
  • the branched linker has four arms.
  • the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided.
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cathepsin-L cleavable linker
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH
  • the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gln- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90.
  • the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cathepsin-L cleavable linker is an un-branched linker.
  • the cathepsin-L cleavable linker is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms.
  • the branched linker has four arms.
  • the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8.
  • the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cathepsin-L cleavable linker
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5.
  • the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Lhr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cathepsin-L cleavable linker is an un-branched linker.
  • the cathepsin-L cleavable linker is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms.
  • the branched linker has four arms.
  • the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8.
  • the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cathepsin-L cleavable linker
  • A is an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90.
  • the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Val- Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Leu-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp- Thr- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cathepsin-L cleavable linker is an un-branched linker.
  • the cathepsin-L cleavable linker is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms.
  • the branched linker has four arms.
  • the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • a cathepsin-L cleavable linker is a cathepsin-L cleavable linker
  • A is an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
  • the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Val- Gln- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Leu-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp- Thr- dipeptide.
  • the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cathepsin-L cleavable linker is an un-branched linker.
  • the cathepsin-L cleavable linker is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms.
  • the branched linker has four arms.
  • the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises Heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Phe-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Phe-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker.
  • the branched linker has two arms. In some embodiments, the branched linker has three arms.
  • the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Phe-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker.
  • the branched linker has two arms. In some embodiments, the branched linker has three arms.
  • the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 1 , 26, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Phe-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90 are provided.
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Phe-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 1 17 are provided.
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Val-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10.
  • the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10. [0539]
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Val-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue.
  • the cleavable linker comprising a -Vai -Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker.
  • the branched linker has two arms. In some embodiments, the branched linker has three arms.
  • the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Val-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker.
  • the branched linker has two arms. In some embodiments, the branched linker has three arms.
  • the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Val-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90 are provided.
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Val-Gln- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 1 17.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided.
  • VH heavy chain variable region
  • VL light chain variable region
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Leu-Gin- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Leu-Gin- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NOVO. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker.
  • the branched linker has two arms. In some embodiments, the branched linker has three arms.
  • the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Leu-Gin- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117.
  • the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker.
  • the branched linker has two arms. In some embodiments, the branched linker has three arms.
  • the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided.
  • the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
  • methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease.
  • the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
  • L is a cleavable linker comprising a -Leu-Gin- dipeptide
  • A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker.
  • the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker.
  • the branched linker has two arms.
  • the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from
  • compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90 are provided.
  • the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2.
  • the average DAR for antibody-payload conjugates in the composition is at least 4.
  • the average DAR for antibody-payload conjugates in the composition is at least 6.
  • the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
  • methods for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90, as described above.
  • the genetic disease or disorder is a neurogenetic disease.
  • the genetic disease or disorder is a musculoskeletal disorder.
  • the genetic disease or disorder is a cardiovascular disease.
  • the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
  • the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping.
  • the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.

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Abstract

The present disclosure provides antibody oligonucleotide conjugates having a 3E10 antibody or an antigen binding fragment thereof conjugated to a single stranded oligonucleotide that may hybridize to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript. Compositions including the antibody oligonucleotide conjugates and methods of using the antibody oligonucleotide conjugates are also provided.

Description

COMPOSITIONS AND METHODS FOR DELIVERING ANTIBODY
OLIGONUCLEOTIDE CONJUGATES FOR EXON SKIPPING
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claim priority to U.S. Provisional Patent Application No. 63/491,859, filed March 23, 2023, U.S. Provisional Patent Application No. 63/515,313, filed July 24, 2023, U.S. Provisional Patent Application No. 63/580,898, filed September 6, 2023, U.S. Provisional Patent Application No. 63/585,840, filed September 27, 2023, and U.S. Provisional Patent Application No. 63/623,890, filed January 23, 2024, the contents of which are hereby incorporated by reference herein, in their entireties, for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on March 23, 2024, is named 127689-5020-WO-SEQ, and is 978,137 bytes in size.
TECHNICAL FIELD
[0003] The present disclosure relates to compositions and methods for treating diseases and disorders with antibody-oligonucleotide conjugates (AOCs).
BACKGROUND
[0004] Several diseases result from genetic mutations that disrupt the reading frame of an encoded polypeptide (e.g., nonsense mutations), resulting in expression of truncated polypeptides or, in some cases, no polypeptide at all. Other diseases result from genetic mutations that introduce new splice sites into a pre-mRNA, or activate cryptic splice sites present in the gene, resulting in aberrant splicing and translation of dysfunctional polypeptides. Exon skipping therapies can be used to treat such diseases. Exon skipping methodologies generally use antisense oligonucleotides (ASO) that bind splice sites in pre-mRNA for an exon containing a deleterious mutation, or bind directly to cryptic splice sites, inducing the splicing machinery to skip over the effective exon or cryptic splice site and generate a mature mRNA that lacks the affected exon, or to ignore the cryptic splice site and generate a full-length mature mRNA. Several antisense oligonucleotides are currently undergoing clinical trials for conditions such as spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), where anti sense-mediated exon skipping can restore the open reading frame and allow the synthesis of partly or wholly functional proteins instead of non- functional ones.
[0005] Duchenne muscular dystrophy, for example, is caused by the absence of dystrophin protein due to mutations in the dystrophin (DMD) gene. The gene encoding the protein contains 79 exons spread out over more than 2 million nucleotides of DNA. Mutations disrupting the reading frame of the protein cause truncation of the translated dystrophin polypeptide, resulting in Duchenne muscular dystrophy. In September 2016, the US Food and Drug Administration (FDA) conditionally approved the first DMD antisense drug, eteplirsen (Exondys 51, SEQ ID NO:208), which was developed to exclude exon 51 in mature DMD mRNA in patients with deleterious mutations in exon 51. Eteplirsen is an antisense oligonucleotide modified with a phosphorodiamidate morpholino oligomer (morpholino or PMO), an antisense chemistry that has been well-established in terms of its safety and effectiveness.
[0006] However, the effectiveness of polynucleotide-based therapies remains questionable. One possible explanation for the poor efficacy of many nucleic acid-based therapies, including exon-skipping therapies, is poor delivery of the therapeutic nucleic acid into the affected tissues. For instance, naked polynucleotides are readily degraded by a host of extracellular nucleases present in human tissues. Further, naked polynucleotides do not readily cross the cell membrane.
[0007] Conventional approaches to overcoming these obstacles include packaging therapeutic polynucleotides into liposomal-based delivery vehicles or recombinant viral particles. However, these approaches present immunological challenges, because the viral capsids and liposomal vehicles are recognized by the host’s immune system.
[0008] The 3E10 antibody is an ideal molecular delivery vehicle due to its efficiency in penetrating into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. 3E10 has not shown any cellular toxicity in vitro or in vivo in studies to date.
SUMMARY
[0009] Given the background above, improved methods are needed for delivering therapeutic antisense oligonucleotides in vivo which are amendable to exon skipping. In particular, there is a need to develop antibody-oligonucleotide conjugates (AOCs), which combine the high precision of siRNA and ASOs, with the deliverability of the 3E10 antibody, taking advantage of both technologies. Advantageously, the present disclosure provides compositions and methods for delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to a single stranded oligonucleotide in vivo that are not reliant upon liposomal or viral vector based nucleic acid delivery.
[0010] In some aspects, the disclosure provides a conjugate of Formula (I):
A-(L-Pr)q
Formula (I), wherein in Formula (I):
A is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of SEQ ID NO:58, CDR2 comprising the amino acid sequence of SEQ ID NO:59, CDR3 comprising SEQ ID NO:60; and a light chain variable region (VL) CDR1 comprising the amino acid sequence of SEQ ID NO:61, CDR2 comprising the amino acid sequence of SEQ ID NO:62, CDR3 comprising the amino acid sequence of SEQ ID NO: 63;
L is a linker;
P is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein; r is an integer from 1 to 4; and q is an integer from 1 to 16.
[0011] In some embodiments, the linker L comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heteroalkylene, optionally substituted heteroarylene, optionally substituted heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -S(O)-, -S(O)2-, -OP(O)ORa-, -OP(O)ORaO-, -P(O)ORaO-, -O-, -CRb2-, -[(CRb 2)1-12O]1-50-, -C(O)-, -C(S)-, -C(=N-OH)-, -C(NRa)-, -C(NH2C1)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -C(O)NRaSO2-, -SO2NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, -S(O)tN(Ra)- (where t is 1 or 2), -N(Ra)S(O>- (where t is 1 or 2), and -XAA-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted alkenyl, optionally substituted fluoroalkenyl, optionally substituted cycloalkenyl, optionally substituted cycloalkenylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -C(0)NRa 2, -CO2Ra, -NRa 2, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted alkenyl, optionally substituted fluoroalkenyl, optionally substituted cycloalkenyl, optionally substituted cycloalkenylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroaryl alkyl; two independent Rb groups or an Ra and an Rb can be joined together to form an optionally substituted cycle; and
-XAA- is an amino acid sequence comprising 1 to 6 amino acid moieties.
[0012] In some embodiments, each amino acid moiety of -XAA- is independently selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Vai), citrulline (Cit), and homocitrulline (HoCit).
[0013] In some embodiments, the linker L comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CRa=CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, -[CH2O]1-18-, -[CH2CH2O]1-18-, -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb 2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, and -XAA-.
[0014] In some embodiments, the linker L comprises one or more groups selected from optionally substituted C1-C16 alkylene, -C=C-, -CRa=CRa-, optionally substituted phenylene, optionally substituted C3-C6 cycloalkylene, -[CH2CH2O]1-16-, -[CH2CH2CH2O]1-16-, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 5- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb 2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)NRa-, -NRaC(O)O-, and -XAA-.
[0015] In some embodiments, the linker L comprises one or more groups selected from -[C(Rb)2]1-16-, -C=C-. -CR — CR'-, -[CH2CH2O]1-16-, -NRa-, -N=CR;|-, -CRa=N-. -S-, -OP(O)ORaO-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)NRa-,
X1, X2, and X3 are independently selected at each occurrence from NRa, N, CRb, S, and O.
[0016] In some embodiments, the linker L is of Formula (L-l):
Formula (L-l), wherein in Formula (L-l):
LA is a connecting moiety through which A is covalently attached to L'; L' is a bond or comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heteroalkylene, optionally substituted heteroarylene, optionally substituted heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -S(O)-, -S(O)2-, -OP(O)ORaO-, -O-, -CRb2-, -[(CRb 2)1-12O]1-50-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2Cl)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -C(O)NRaSO2-, -SO2NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, -S(O)tN(Ra)-, -N(Ra)S(O)t-, and -XAA-; and
Lp is a connecting moiety through which P is covalently attached to L'.
[0017] In some embodiments, the linker L comprises at least one cleavable moiety. In some embodiments, the cleavable moiety comprises an acid-labile moiety, a reducibly-labile moiety, or an enzymatically-labile moiety. In some embodiments, the cleavable moiety comprises one or more groups selected from: wherein: each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl.
[0018] In some embodiments, the cleavable moiety comprises the reducibly-labile moiety -S-S-.
[0019] In some embodiments, the linker L is of Formula (L-10):
Formula (L-10), wherein in Formula (L-10): LA is selected from a bond, -NRa'-, and -S-;
L1 is a bond or comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CR — CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, -[CH2O]1-18-, -[CH2CH2O]1-18-, -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb 2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, and -XAA-;
Lc is selected from an acid-labile moiety, a reducibly-labile moiety, and an enzymatically- labile moiety;
L2 is a bond or comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CR — CR1-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene. -[CH2O]1-18-, -[CH2CH2O]1-18-, -[CH2CH2CH2O]1-18 -, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, and -XAA-;
LP is selected from a bond, -NRa-, -S-, and -O-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each Ra’ is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -C(0)NRa 2, -CO2Ra, -NRa 2, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycloalkyl, or optionally substituted heteroaryl; and
-XAA- is an amino acid sequence comprising 1 to 4 amino acid moieties.
[0020] In some embodiments, Lc is selected from: wherein: each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl.
[0021] In some embodiments, Lc is -S-S-.
[0022] In some embodiments, the linker L is of Formula (L-l 1):
Formula (L-l 1), wherein in Formula (L-l 1):
LA is selected from a bond, -NH-, and -S-;
L1 is a bond or comprises one or more groups selected from -[C(Rb)2]1-16-, -C=C-, - CRa= CRa-, -[CH2CH2O]1-16-, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-, -NRaC(O)O-,
L2 is a bond or comprises one or more groups selected from -[C(Rb)2]i i6-, -C=C-,
-CRa=CRa-, -[CH2CH2O]1-16-, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -0-, -C(0)-, -C(0)0-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-, -NRaC(O)O-,
Lp is selected from a bond, -NRa -, and -O-; each Ri is independently selected from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or both Ri groups are taken together to form optionally substituted cycloalkyl; each R2 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or both R2 groups are taken together to form optionally substituted cycloalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each Ra is independently selected at each occurrence from hydrogen and optionally substituted alkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl; and
-XAA- is an amino acid sequence comprising 2 to 4 amino acid moieties.
[0023] In some embodiments, the linker L is of Formula (L-12):
Formula (L-12), wherein in Formula (L-12):
LA is selected from a bond and -NH-;
L1' comprises one or more groups selected from -[C(Rb)2]1-10-, -[CH2CH2O]1-10-, -NRa-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-, L2’ comprises one or more groups selected from -[C(Rb)2]1-10-, -[CH2CH2O]I-IO-, -NRa-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-,
Lp is selected from a bond and -NRa’-; each Ri is independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or both Ri groups are taken together to form optionally substituted C3-C6 cycloalkyl; each R2 is independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or both R2 groups are taken together to form optionally substituted C3-C6 cycloalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 fluoroalkyl, and optionally substituted C3-C6 cycloalkyl; each Ra' is independently selected at each occurrence from hydrogen and optionally substituted C1-C6 alkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 fluoroalkyl, and optionally substituted C3-C6 cycloalkyl; or two independent Rb groups attached to the same carbon atom are taken together to form optionally substituted C3-C6 cycloalkyl; and
-XAA- is an amino acid sequence comprising 2 or 3 amino acid moieties.
[0024] In some embodiments, at least one Ri or R2 is other than hydrogen. In some embodiments, at least one Ri is an optionally substituted C1-C8 alkyl. In some embodiments, each Ri is independently an optionally substituted C1-C8 alkyl. In some embodiments, at least one R2 is an optionally substituted C1-C8 alkyl. In some embodiments, each R2 is independently an optionally substituted C1-C8 alkyl.
[0025] In some embodiments, the linker L is selected from:
[0027] In some embodiments, the linker is a cleavable linker.
[0028] In some embodiments, the linker L is of Formula (L-20):
Formula (L-20), wherein in Formula (L-20):
LA is selected from a bond, -NRa'-, and -S-; L3 is a bond or comprises one or more groups selected from -[C(Rb)2]1-8-, -NRa-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2CI)-, -C=C-, - CRa= CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, optionally substituted 5- to 18-membered heteroarylene, and optionally substituted 3- to 20-membered heterocycloalkylene;
Lx comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CRa=CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, -[CJ OJ1-18-, -[CJLCI OJ1-18-, -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -S-, -O-, -CRb 2-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2C1)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, and -NRaC(O)-;
L4 is a bond or comprises one or more groups selected from -[C(Rb)2]1-8-, -NRa-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2Cl)-, -C=C-, -CRa=CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, optionally substituted 5- to 18-membered heteroarylene, and optionally substituted 3- to 20-membered heterocycloalkylene;
Lp is selected from a bond, -NRa -, -S-, and -O-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl; and each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -C(O)NRa 2, -CO2Ra, -NRa 2, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycloalkyl, or optionally substituted heteroaryl.
[0029] In some embodiments, the linker L is of Formula (L-21):
Formula (L-21), wherein in Formula (L-21):
LA is selected from a bond and -NH-;
LX comprises one or more groups selected from optionally substituted -[C(Rb)2]1-16-, -C=C-, -CRa=CRa-, -[CH2CH2CH2O]1-16-, -NRa-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-,
LP is selected from a bond and -NRa’-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each Ra is independently selected at each occurrence from hydrogen and optionally substituted C1-C6 alkyl; and each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted Ci-Cx alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl.
[0030] In some embodiments, the linker L is of Formula (L-22a) or Formula (L-22b):
Formula (L-22b), wherein in Formulas (L-22a) and (L-22b):
LA is selected from a bond and -NH-;
Lx comprises one or more groups selected from optionally substituted -[C(Rb)2]1-10-,
-C=C-, -CR — CR1-, -[CH2CH2CH2O]1-10-, -NRa-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-,
Lp is selected from a bond and -NRa'-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each Ra' is independently selected at each occurrence from hydrogen and optionally substituted C1-C6 alkyl; and each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl.
[0031] In some embodiments, the linker L is selected from:
[0032] In some embodiments, the linker is a non-cleavable linker.
[0034] In some embodiments, each amino acid moiety of -XAA- is independently selected from alanine (Ala), arginine (Arg), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), valine (Vai), citrulline (Cit), and homocitrulline (HoCit). In some embodiments, each amino acid moiety of -XAA- is independently selected from alanine (Ala), glycine (Gly), lysine (Lys), phenylalanine (Phe), valine (Vai), and citrulline (Cit). In some embodiments, the amino acid sequence -XAA- is selected from -Val-Cit-, -Cit-Val-, -Vai-Ala-, -Ala-Val-, -Phe-Lys-, -Lys-Phe-, -Ala-Ala-, -Val-Val-, -Gly-Gly-, -Ala-Ala-Ala-, -Gly-Gly-Gly-, Gly-Gly-Phe-Gly-(SEQ ID NO: 1032) , -Gly-Phe-Gly-Gly-(SEQ ID NO: 1033),
-Gly-Gly-Gly-Phe-(SEQ ID NO: 1034), -Phe-Gly-Gly-Gly-(SEQ ID NO: 1035), and -Gly-Gly-Gly-Gly-(SEQ ID NO: 1036). In some embodiments, the amino acid sequence -XAA- is selected from -Val-Cit-, -Cit-Val-, -Val-Ala-, -Ala-Val-, -Phe-Lys-, -Lys-Phe-, -Ala-Ala-, -Val-Val-, -Gly-Gly-, -Ala-Ala-Ala-, and -Gly-Gly-Gly-.
[0035] In some embodiments, the single stranded oligonucleotide P is a phosphorodiamidate morpholino oligonucleotide or an antisense oligonucleotide. In some embodiments, the single stranded oligonucleotide P is delivered into a muscle cell. In some embodiments, the single stranded oligonucleotide P induces skipping of exon 23 of the DMD gene.
[0036] In some embodiments, the phosphorodiamidate morpholino oligonucleotide comprises the sequence 5'-C6 Amino-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO:408). In some embodiments, the antisense oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 1045, SEQ ID NOs: 158-222, SEQ ID NO:395-405, and SEQ ID NO:410-988. In some embodiments, the antisense oligonucleotide is a peptide nucleic acid (PNA) oligonucleotide. In some embodiments, the peptide nucleic acid (PNA) oligonucleotide comprises the sequence (C)-3’-TAAAGTCCATTCGGCTCCAAACCGG-C6 Amino-5’(N) (SEQ ID NO:409). In some embodiments, the single stranded oligonucleotide P comprises at least from about 10 to about 30 nucleotides in length.
[0037] In some embodiments, the truncated protein modulates muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. [0038] In some embodiments, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:9, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, CDR3 comprises SEQ ID NO: 11 ; and the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 15, CDR2 comprises the amino acid sequence of SEQ ID NO:4, CDR3 comprises the amino acid sequence of SEQ ID NO:5.
[0039] In some embodiments, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:29, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, CDR3 comprises SEQ ID NO: 11; and the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 15, CDR2 comprises the amino acid sequence of SEQ ID NO:26, CDR3 comprises the amino acid sequence of SEQ ID NO:5.
[0040] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO:21 and a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 14.
[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence of SEQ ID NO:20 and a full length heavy chain (HC) comprising an amino acid sequence of SEQ ID NO: 13.
[0042] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VL-H1 (SEQ ID NO:85), 3E10-VL-H2 (SEQ ID NO:86), 3E10-VL-H3 (SEQ ID NO:87), 3E10-VL-H4 (SEQ ID NO:88), 3E10-VL-H5 (SEQ ID NO: 89), and 3E10-VL-H6 (SEQ ID NO: 90); and a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-H1 (SEQ ID NO:64), 3E10-VH-H2 (SEQ ID NO:65), 3E1O-VH-H3 (SEQ ID NO:66), 3E10-VH-H4 (SEQ ID NO:67), 3E1O-VH-H5 (SEQ ID NO:68), 3E10-VH-H6 (SEQ ID NO:69), and 3E10-VH-H7 (SEQ ID NO:70).
[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence selected from the group consisting of 3E10-VL-H1 (SEQ ID NO:85), 3E10-VL-H2 (SEQ ID NO:86), 3E10-VL-H3 (SEQ ID NO:87), 3E10-VL-H4 (SEQ ID NO:88), 3E10-VL-H5 (SEQ ID NO:89), and 3E10-VL-H6 (SEQ ID NO:90); and a heavy chain variable domain (VH) comprising an amino acid sequence selected from the group consisting of 3E10-VH-H1 (SEQ ID NO:64), 3E10-VH-H2 (SEQ ID NO:65), 3E10-VH-H3 (SEQ ID NO:66), 3E10-VH-H4 (SEQ ID NO:67), 3E10-VH-H5 (SEQ ID NO:68), 3E10-VH-H6 (SEQ ID NO:69), and 3E10-VH-H7 (SEQ ID NO:70).
[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL / VH pair selected from the group consisting of (a) VL1 (SEQ ID NO:85) and VH1 (SEQ ID NO:64), (b) VL1 (SEQ ID NO:85) and VH2 (SEQ ID NO:65), (c) VL1 (SEQ ID NO:85) and VH3 (SEQ ID NO:66), (d) VL1 (SEQ ID NO:85) and VH4 (SEQ ID NO:67), (e) VL2 (SEQ ID NO:86) and VH1 (SEQ ID NO:64), (f) VL2 (SEQ ID NO:86) and VH2 (SEQ ID NO:65), (g) VL2 (SEQ ID NO:86) and VH3 (SEQ ID NO:66), (h) VL2 (SEQ ID NO:86) and VH4 (SEQ ID NO:67), (i) VL3 (SEQ ID NO:87) and VH1 (SEQ ID NO:64), (j) VL3 (SEQ ID NO:87) and VH2 (SEQ ID NO:65), (k) VL3 (SEQ ID NO:87) and VH3 (SEQ ID NO:66), (1) VL3 (SEQ ID NO:87) and VH4 (SEQ ID NO:67), (m) VL4 (SEQ ID NO:88) and VH1 (SEQ ID NO:64), (n) VL4 (SEQ ID NO:88) and VH2 (SEQ ID NO:65), (o) VL4 (SEQ ID NO:88) and VH3 (SEQ ID NO:66), (p) VL4 (SEQ ID NO:88) and VH4 (SEQ ID NO:67), (q) VL5 (SEQ ID NO:89) and VH5 (SEQ ID NO:68), (r) VL5 (SEQ ID NO:89) and VH6 (SEQ ID NO:69), (s) VL6 (SEQ ID NO:90) and VH5 (SEQ ID NO:68), and (t) VL6 (SEQ ID NO:90) and VH6 (SEQ ID NO:69).
[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising 3E10-VL-H6 (SEQ ID NOVO) and a heavy chain variable domain (VH) comprising 3E10-VH-H6 (SEQ ID NO:69).
[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising the amino acid sequence (DIQMTQSPSSLSASLGDRATITCRASKTVSTSSYSYMHWYQQKPGQPPKLLIKYASYLE SGVPSRFSGSGSGTDFTLTISSLQPEDAATYYCQHSREFPWTFGGGTKVEIK) (SEQ ID NO: 117) and a heavy chain variable domain (VH) comprising the amino acid sequence (EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSYISSGSSTI YYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRGLLLDYWGQGTTVTVS S) (SEQ ID NO: 105).
[0047] In some aspects, the present disclosure provides a method for treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a conjugate of the disclosure to the subject. [0048] In some aspects, the present disclosure provides a method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising administering a therapeutically effective amount of a conjugate of the disclosure to the subject.
[0049] In another aspect, the disclosure provides a method for delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to a single stranded oligonucleotide to a tissue of a subject in vivo, the method including parenterally administering a pharmaceutical composition, as described herein, to the subject. In some embodiments, the antisense oligonucleotide is for treating a disease or disorder including, but not limited to a skeletal muscle disorder, a neurogenetic disease, a cardiovascular disease, a metabolic disease, or a lung disorder for which a known disease-causing mutation.
[0050] In some embodiments of the methods and compositions described herein, the 3E10 antibody or antigen-binding fragment thereof includes (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL- CDR1 (SEQ ID NO: 9), (b) a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), (c) a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRla (SEQ ID NO: 16), (e) a VH CDR2 comprising the amino acid sequence of 3E10- VH-CDR2 (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of 3E10- VH-CDR3 (SEQ ID NO: 5).
[0051] In some embodiments of the methods and compositions described herein, the 3E10 antibody or antigen-binding fragment thereof includes (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR1 (SEQ ID NO: 9), (b) a VL CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR2 (SEQ ID NO: 10), (c) a VL CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR3 (SEQ ID NO: 11), (d) a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDRla (SEQ ID NO: 3), (e) a VH CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH- CDR2 (SEQ ID NO: 4), and (f) a VH CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR3 (SEQ ID NO: 5).
[0052] In some embodiments of the methods and compositions described herein, the 3E10 antibody or antigen-binding fragment thereof includes (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL- CDRlm (SEQ ID NO: 61), (b) a VL CDR2 comprising the amino acid sequence of 3E10-VL- CDR2m (SEQ ID NO: 62), (c) a VL CDR3 comprising the amino acid sequence of 3E10-VL- CDR3m (SEQ ID NO: 63), (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO: 58), (e) a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO: 59), and (f) a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO: 60).
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0054] Figure 1 (SEQ ID NOs: 1-12) illustrates amino acid sequences for the parent 3E10 monoclonal antibody.
[0055] Figures 2A (SEQ ID NOs: 13-25), 2B (SEQ ID NOs:26-30), and 2C (SEQ ID NOs:31-33) illustrate amino acid sequences for the D3 IN variant (Figure 2A), other CDR variants (Figure 2B), and additionally contemplated CDR variants (Figure 2C) of the 3E10 monoclonal antibody, in accordance with some embodiments of the present disclosure.
[0056] Figure 3 (SEQ ID NOs:34-57) illustrates example charge-conserved CDR variants of the 3E10 monoclonal antibody, in accordance with various embodiments of the present disclosure.
[0057] Figure 4 (SEQ ID NOs: 58-63) illustrates example CDR variants containing a combination of amino acid substitutions, charged-conserved amino acid substitutions, and rationally-designed amino acid substitutions of the 3E10 monoclonal antibody, in accordance with various embodiments of the present disclosure. [0058] Figure 5 (SEQ ID NOs: 103-112) illustrates a sequence alignment of examples of humanized 3E10 heavy chain variable regions, with CDRs underlined as indicated.
[0059] Figure 6 (SEQ ID NOs: 113-121) illustrates a sequence alignment of examples of humanized 3E10 light chain variable regions, with CDRs and putative nuclear localization signals (NLS) underlined as indicated.
[0060] Figures 7A, 7B, 7C, 7D, and 7E (SEQ ID NOs: 122-137) collectively illustrate a sequence alignment of example of humanized di-scFv constructs of the 3E10 monoclonal antibody.
[0061] Figure 8 illustrates 3E10 (V66)-phosphorodiamidate morpholino oligomer (PMO) conjugates using three separate linker chemistries, maleimide, SATA-SAPP, and DBCO.
[0062] Figures 9 illustrates 3E10-phosphorodiamidate morpholino oligomer (PMO) conjugates demonstrating dose-dependent exon skipping in vitro.
[0063] Figure 10 illustrates single dose exon skipping in Duchenne muscular dystrophy (DMD) using a 3E10-phosphorodiamidate morpholino oligomer (PMO) conjugate. Abbreviations shown: Tibialis anterior (T), Gastrocnemius (G), Quadricep (Q), Deltoid (D), and Heart (H).
[0064] Figures 11A and 11B illustrate electrostatic surface potential renderings of a molecular model of a 3E10-scFv construct, revealing a putative Nucleic Acid Binding pocket (NAB1). Figure 11A additionally shows predicted structural and electrostatic potential changes induced by amino acid substitutions at residue HC CDR1 residue 31. Figure 1 IB is an illustration of molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues highlighted by punctate dots.
[0065] Figure 11C illustrates mapping of the putative nucleic acid binding pocket, as identified by the molecular modeling shown in Figures 11 A and 1 IB, onto the amino acid sequence of the 3E10-scFv construct.
[0066] Figure 12 shows the study design for confirmatory single dose exon skipping by 3E10 (V66) in mdx mice.
[0067] Figure 13 shows gel electrophoresis analysis of follow-on single dose IV study performed to confirm DMD functional skipping in mdx mice with 3E10 (V66) non-cleavable linker-PMO conjugate. [0068] Figure 14A illustrates the results of DMD exon skipping detected across skeletal and heart muscle in mdx mice receiving non-cleavable and disulfide cleavable 3E10 (V66) PMO conjugates.
[0069] Figure 14B illustrates single low-dose administration of 3E10 (V66) - PMO conjugates with cleavable and non-cleavable linkers in selected tissues in mdx mice.
[0070] Figure 15 illustrates nucleotide sequences (SEQ ID NO: 1045, 405, and 1041-1044) for the oligomers designed to cause skipping of exon 23 in dystrophin (DMD) RNA. K denotes lysine residues on PNA; superscript O denotes 2’OMe modifications; superscript F denotes 2’fluoro modifications; superscript L denotes locked nucleic acid (LNA) modifications.
[0071] Figure 16 illustrates amino acid sequences of humanized 3E10 variable heavy (3E10-VH) domains (SEQ ID NOs:64-70), in accordance with various embodiments of the present disclosure.
[0072] Figure 17 illustrates amino acid sequences of mature humanized 3E10 heavy chains (3E10-HC) lacking a signal peptide (SEQ ID NOs:71-77), in accordance with various embodiments of the present disclosure.
[0073] Figure 18 illustrates amino acid sequences of humanized 3E10 heavy chains (3E10-HC) (SEQ ID NOs:78-84), in accordance with various embodiments of the present disclosure.
[0074] Figure 19 illustrates amino acid sequences of humanized 3E10 variable light (3E10-VL) domains (SEQ ID NOs:85-90), in accordance with various embodiments of the present disclosure.
[0075] Figure 20 illustrates amino acid sequences of mature humanized 3E10 light chains (3E10-LC) lacking a signal peptide (SEQ ID NOs:91-96), in accordance with various embodiments of the present disclosure.
[0076] Figure 21 illustrates amino acid sequences of humanized 3E10 light chains (3E10- LC) (SEQ ID NOs:97-98, 100-102, and 1045), in accordance with various embodiments of the present disclosure.
[0077] Figure 22 illustrates western blots of ENT2 protein expression in selected human and mouse healthy tissues. [0078] Figure 23 illustrates the internalization of a labeled 3E10 (V66) - PMO conjugate in C2C12 muscle myotubes and A427 tumor cells.
[0079] Figure 24 illustrates the experimental design for single and repeat dose studies with 3E10 (V66) - PMO conjugates measuring exon skipping in mdx mice.
[0080] Figures 25A-25C illustrate the durability over time of the 3E10 (V66) - PMO conjugate administration for exon skipping in deltoid and quadriceps (Figure 25A), in tibialis anterior and gastrocnemius (Figure 25B), and in heart and diaphragm (Figure 25C).
[0081] Figure 26 illustrates single dose administration of the 3E10 (V66) - PMO conjugate exhibits dystrophin protein restoration.
[0082] Figures 27A and 27B illustrate dystrophin restoration after administration of the 3E10 (V66) - PMO conjugate in selected tissues on Day 14 (Figure 27A) and on Day 28 (Figure 27B).
[0083] Figure 28A illustrates the process for quantifying attachment sites for Lys-azide conjugation intermediates of 3E10-D31N monoclonal antibody (V66), illustrating peptides KVEPK (SEQ ID NO: 1022) and K*VEPK (SEQ ID NO: 1023) released by proteolysis.
[0084] Figure 28B shows the mass spectroscopy results of mapped Lys-azide conjugation intermediates of 3E10-D31N monoclonal antibody (V66) after proteolysis.
[0085] Figure 28C shows an alignment of chimeric 3E10-D31N variable heavy and variable light chains with the corresponding sequences of a humanized 3E10-D31N antibody (V66), in accordance with various embodiments of the present disclosure. The consensus sequence for the VH is SEQ ID NO: 1024. The 3E10-D31N-VH sequence is SEQ ID NO: 1025. The 3E10- D31N-VH6 sequence is SEQ ID NO:69. The consensus sequence for the VL is SEQ ID NO: 1026. The 3E10-D31N-VL sequence is SEQ ID NO:8. The 3E10-D31N-VL6 sequence is SEQ ID NO:90.
[0086] Figures 29A and 29B collectively illustrate improved cellular internalization of 3E10-D31N monoclonal antibody (V66) oligonucleotide conjugates in A427 cells utilizing trasglutaminase-mediated enzymatic conjugation.
[0087] Figure 30A shows improved exon skipping utilizing transglutaminase-mediated enzymatic conjugation. Figure 30B shows varying the length of the PEG linkers (e.g., PEG4, PEG8, and PEG12) did not significantly impact exon skipping. Figure 30C shows enhanced exon- skipping of cleavable linkers, protease Cathepsin-B and SPDMV (disulfide) compared to non- cleavable linkers with transglutaminase-mediated enzymatic conjugation.
[0088] Figure 31A show a di-methyl-hindered disulfide cleavable linker and protease cleavable linker have greater stability over a single methyl-hindered disulfide linker in mouse serum. Figure 31B show a data table indicating the panel of antibody-oligonucleotide conjugates (AOCs) tested and their oligonucleotide to antibody ratios (OAR or DAR).
[0089] Figure 32 shows a summary table of the in vitro performance of several antibody- oligonucleotides conjugates utilizing lysine and transglutaminase-mediated conjugation.
[0090] Figure 33 shows levels of exon skipping of DMD preRNA at day 7 post-dose in mdx mice that were administered a 3E10 AOC with a noncleavable linker. Doses shown for each muscle group are, from left to right, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg.
[0091] Figure 34 shows quantification of delivered PMO at day 7 post-dose as determined by hybridization ELISA in diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker. Doses shown for each muscle group are, from left to right, vehicle, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg.
[0092] Figure 35A shows quantification of delivered PMO at day 14 post-dose as determined by hybridization ELISA in diaphragm, quadriceps, and gastrocnemius tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker. Figure 35B shows quantification of delivered PMO at day 14 post-dose as determined by hybridization ELISA in tibialis anterior, deltoid, and heart tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker.
[0093] Figure 36 shows levels over time of exon skipping of DMD preRNA in mdx mice that were administered a 3E10 AOC with a noncleavable linker.
[0094] Figure 37 shows quantification of delivered PMO at day 10 post-dose as determined by hybridization ELISA in diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker. Cohorts shown for each muscle group are, from left to right, vehicle, PMO30, and AOC30.
[0095] Figure 38 shows levels of exon skipping of DMD preRNA at day 10 post-dose in mdx mice that were administered a 3E10 AOC with a noncleavable linker compared to PMO alone (PMO23) or vehicle control.
[0096] Figure 39 shows levels of dystrophin protein restoration at day 28 post-dose in heart, diaphragm, tibialis anterior (TA), and quadriceps (Q) muscle tissue samples from mdx mice that were administered a 3E10 AOC with a noncleavable linker. Doses shown for each muscle group are, from left to right, 18 mg/kg, 30 mg/kg, and 42 mg/kg.
[0097] Figures 40A-40C show day 28 post-dose immunofluorescent detection of dystrophin in the diaphragm of WT mice (Figure 40A), mdx mice (Figure 40B), and mdx mice that were administered a 3E10 AOC with a noncleavable linker (Figure 40C).
[0098] Figure 41 illustrates a 3E10 (V66)-phosphorodiamidate morpholino oligomer (PMO) conjugate having a Cathepsin B cleavable linker.
[0099] Figure 42 shows levels of exon skipping of DMD preRNA at day 14 post-dose in mdx mice that were administered vehicle control, a Tg-noncleavable 3E10 AOC, and a Tg- cleavable 3E10 AOC. The muscle groups shown, from left to right, are, diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H).
[0100] Figure 43 shows levels of exon skipping of DMD preRNA at day 14 post-dose in mdx mice that were administered vehicle control, a Lys-noncleavable 3E10 AOC, and a Tg- noncleavable 3E10 AOC. The muscle groups shown, from left to right, are, diaphragm, quadriceps, gastrocnemius, tibialis anterior, deltoid, and heart.
[0101] Figure 44 shows levels of exon skipping of DMD preRNA at day 28 post-dose in mdx mice that were administered a noncleavable 3E10 AOC and a disulfide cleavable 3E10 AOC. The muscle groups shown, from left to right, are, diaphragm, tibialis anterior (TA), heart, deltoid (Delt), gastrocnemius (Gastroc), and quadriceps (Quad).
[0102] Figure 45 shows day 28 post-dose levels of dystrophin protein restoration in diaphragm, tibialis anterior, quadriceps, and deltoid muscle tissue samples from mdx mice that were administered a noncleavable 3E10 AOC and a disulfide cleavable 3E10 AOC. [0103] Figure 46 shows day 28 post-dose quantification of dystrophin protein distribution in diaphragm, heart, and tibialis anterior (TA) of mdx mice that were administered a noncleavable 3E10 AOC and a disulfide cleavable 3E10 AOC.
[0104] Figure 47 shows in situ hybridization analysis of PMO distribution in the deltoid muscle of a mouse that was administered a noncleavable 3E10 AOC, at 20x magnification.
[0105] Figure 48 shows in situ hybridization analysis of PMO distribution in the deltoid muscle of a mouse that was administered a noncleavable 3E10 AOC, at 40x and lOOx magnification.
[0106] Figure 49 shows in situ hybridization analysis of PMO distribution in the heart muscle of mice that were administered vehicle control, a cleavable 3E10 AOC, or a noncleavable 3E10 AOC, at 40x magnification.
[0107] Figure 50 shows quantification at day 7 post-dose of delivered PMO as determined by hybridization ELISA in deltoid, diaphragm, heart, and tibialis anterior muscle tissue samples from mice that were administered vehicle, PMO alone, PPMO alone, or a disulfide cleavable 3E10 AOC. Cohorts shown for each muscle group are, from left to right, vehicle, PMO, PPMO, and AOC.
[0108] Figure 51 shows in situ hybridization analysis of PMO distribution in the deltoid muscle of mice that were administered PMO alone, PPMO alone, or a disulfide cleavable 3E10 AOC, at 4x and 40x magnification.
[0109] Figure 52 illustrates various linker designs for generating 3E10 AOCs, i.e., a phosphatase cleavable linker, a glucuronidase cleavable linker, an SPDMV disulfide cleavable linker, and a SPDB disulfide cleavable linker.
[0110] Figures 53A and 53B show levels of exon skipping of DMD preRNA in mdx mice that were administered 3E10 AOCs having (Lys)-DBCO-PEG8-PMO, (Lys)-DBCO-PEG4- SPDMV-PMO, or (Lys)-DBCO-PEG8-PO4-PAB-PMO linkers (Figure 48A), or (Tg)-DBCO- PEG8-PMO, (Tg)-DBCO-PEG4-SPDMV-PMO, or (Tg)-CathB-PEG8-PMO linkers (Figure 48B).
[0111] Figure 54A illustrates the design of a site-specific DAR 4 3E10 AOC transglutaminase conjugate comprising a hindered disulfide SPDMV linker (Tg-SPDP). Figure 54B shows mass spectrometry data for the site-specific DAR 4 3E10 AOC transglutaminase conjugate comprising a hindered disulfide SPDMV linker (Tg-SPDP). Figure 54C shows levels of exon skipping of DMD preRNA in mdx mice that were administered a noncleavable 3E10 AOC (Tg-Peg8), or three cleavable 3E10 AOCs (Tg-SPDMV, Tg-CathB, and Tg-SPDP) .
DETAILED DESCRIPTION
L _ Introduction
[0112] The present disclosure provides compositions, conjugates, and methods for delivering therapeutic polynucleotides, e g., antisense oligonucleotides, that are amendable to exon skipping in vivo, and that are not reliant upon the conventional viral-based or liposomal-based delivery methodologies associated with difficult and costly production, limited packaging capacity, and adverse immunological events. In some aspects, described in greater detail below, these compositions and methods are based on the covalent attachment of a 3E10 antibody or an antigen binding fragment thereof to a single stranded oligonucleotide, forming a conjugate, increasing the in vivo effectiveness of these complexes. In some embodiments, the methods and compositions find particular use for the treatment of genetic diseases and disorders, including neurogenetic diseases, musculoskeletal disorders, cardiovascular diseases, metabolic diseases, cancers, lung disorders, and other diseases that can be benefitted by exon-skipping therapies. For instance, described herein are compositions comprising a conjugate of (i) a 3E10 antibody or antigen-binding fragment thereof, and (ii) an antisense oligonucleotide, as well as methods for using such compositions for the treatment of diseases and disorders, including neurogenetic diseases, musculoskeletal disorders, cardiovascular diseases, metabolic diseases, cancers, lung disorders, and other diseases that can be benefitted by exon-skipping therapies described herein.
[0113] The studies described herein demonstrate that conjugates comprising a cell- penetrating and nucleic acid-binding antibody (3E10) conjugated via a linker to a therapeutic antisense oligonucleotides can effectively cause exon skipping in vitro and in vivo. For example, Example 2 demonstrates that both stable (non-cleavable) and cleavable 3E10 (V66) conjugates to a phosphorodi ami date morpholino oligomer (PMO) targeting exon 23 of DMD caused dose- dependent exon-skipping in differentiated C2C12 myotubules. Examples 4-6 and 9 demonstrate that V66-PMO conjugates were able to cause exon-skipping in an mdx DMD mouse model. Example 7 demonstrates that there is high ENT2 protein expression in mouse and human muscle, as well as in the heart and diaphragm tissue. Examples 8 and 11 demonstrate that V66-PMO conjugates are internalized in C2C12 muscle myotubes and A427 tumor cells. Example 9 further demonstrates that a V66-PMO conjugate was able to restore dystrophin expression in muscle tissues in mdx mice models of DMD.
[0114] In some embodiments, the advantageous properties of the compositions and methods described herein are based, at least in part, on the substantial targeting and delivery of exon-skipping oligonucleotides to muscle tissues when conjugated to 3E10 antibodies and antigen- binding fragments thereof. For instance, as described in Example 13, and exemplified in Figure 34, high levels of tissue PMO delivery was observed in diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue at 7 days post- treatment. Moreover, the delivery demonstrated a dose-dependency in all tissues, including the heart. Delivery of the PMO exon skipping oligo to these muscle tissues was approximately 100- fold higher when conjugated to 3E10 than when injected alone without 3E10, as described in Example 14 and exemplified in Figure 37.
[0115] In some embodiments, the advantageous properties of the compositions and methods described herein are based, at least in part, on the persistent localization of exon skipping oligonucleotides to when conjugated to 3E10 antibodies and antigen-binding fragments thereof. For instance, as described in Example 13, and exemplified in Figures 35A-35B, high levels of tissue PMO localization to diaphragm (Di), quadriceps (Q), gastrocnemius (G), tibialis anterior (TA), deltoid (D), and heart (H) muscle tissue persisted 14-days post-treatment. Moreover, the localization followed a dose-dependency.
[0116] In some embodiments, the advantageous properties of the compositions and methods described herein are based, at least in part, on the persistent exon skipping induced by oligonucleotides when conjugated to 3E10 antibodies and antigen-binding fragments thereof. For instance, as described in Example 14, and exemplified in Figure 36, exon skipping in was increased in diaphragm (Di), tibialis anterior (TA), and heart (H) muscle tissue at day 20 post-injection, relative to exon skipping at day 10 post-injection.
[0117] In some embodiments, the advantageous properties of the compositions and methods described herein are based, at least in part, on the even distribution of exon-skipping oligos across muscle tissues when conjugated to 3E10 antibodies and antigen-binding fragments thereof. For instance, as described in Example 16, and exemplified in Figures 47-49, PMO oligo was evenly-distributed across deltoid and heart tissues when administered conjugated to 3E10. Moreover, as described in Example 17 and exemplified in Figure 51, significantly more PMO exon-skipping oligonucleotide was delivered to the deltoid, and translocated to the nucleus of muscle cells, when administered conjugated to 3E10 than when administered alone (compared to both PMO and PPMO oligonucleotides).
[0118] Accordingly, in one aspect, the disclosure provides a conjugate of Formula (I):
A-(L-Pr)q Formula (I), wherein in Formula (I): A is an antibody, antigen-binding fragment thereof or antigen-binding fragment thereof comprising a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of SEQ ID NO:58, CDR2 comprising the amino acid sequence of SEQ ID NO:59, CDR3 comprising SEQ ID NO:60; and a light chain variable region (VL) CDR1 comprising the amino acid sequence of SEQ ID NO:61, CDR2 comprising the amino acid sequence of SEQ ID NO:62, CDR3 comprising the amino acid sequence of SEQ ID NO:63; L is a linker; P is an oligonucleotide capable of hybridizing to a pre-mRNA transcript, wherein the oligonucleotide induces exon skipping in the pre-mRNA transcript; r is an integer from 1 to 4; and q is an integer from 1 to 16.
[0119] Similarly, in one aspect, the disclosure provides a method for inducing exon skipping in a target tissue in a subject by administering a conjugate of Formula (I). In some embodiments, the target tissue is a muscle tissue. In some embodiments, the target tissue is diaphragm tissue. In some embodiments, the target tissue is quadricep tissue. In some embodiments, the target tissue is gastrocnemius tissue. In some embodiments, the target tissue is tibialis anterior tissue. In some embodiments, the target tissue is deltoid tissue. In some embodiments, the target tissue is heart muscle tissue.
[0120] In some embodiments, the disclosure provides a method for treating a disorder in a subject in need thereof by administering a therapeutically effective amount of a composition comprising a conjugate of Formula (I) to the subject. In some embodiments, the disorder is a muscle disorder. In some embodiments, the muscle disorder is a muscular dystrophy. In some embodiments the muscular dystrophy is Duchenne muscular dystrophy (DMD). II. Definitions
[0121] The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting.
[0122] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term “and/or” as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items. Unless the context requires otherwise, it will be further understood that the terms “includes,” “comprising,” or any variation thereof, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” Additionally, where the terms “comprising,” “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, alternatives reciting “consisting of’ or “consisting essentially of’ are intended to be encompassed within such disclosures.
[0123] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
[0124] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%.
[0125] As used herein, the term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term “antibody,” as used herein, is used in the broadest sense and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and antibody fragments (such as Fab, Fab’, F(ab’)2, Fv fragments, scFv molecules), and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as they exhibit one or more of the desired biological activities. In embodiments, “desired biological activity” of an antibody refers to the ability of the antibody to bind to its target antigen, e.g., a nucleic acid, e.g., DNA. In embodiments, “desired biological activity” can further include antibody binding to its target antigen and resulting in a measurable biological response which can be measured in vitro or in vivo. Such activity can be antagonistic or agonistic. In embodiments, “desired biological activity” of an antibody refers to the ability of the antibody to bind to a target, e.g., nucleic acid molecules. In embodiments, “desired biological activity” of an antibody refers to the ability of the antibody to bind to a cellular receptor, e.g., ENT2. In embodiments, “desired biological activity” of an antibody refers to the ability of the antibody to be internalized by a target cell. “Target antigen,” as used herein, refers to the molecule that is bound specifically by the antigen-binding domain comprising the variable regions of a given antibody. The term “specifically binds” refers to the binding of an antibody to its cognate antigen (e.g., a nucleic acid, e.g., DNA) while not significantly binding to other antigens.
[0126] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses or isotypes, e.g., IgG1, IgG2, IgG3 , IgG4 , IgA1, and IgA . “Isotype,” as used herein, refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, y, s, y, and p. respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). It should be understood that antibodies disclosed herein can also comprise hybrids of isotypes and/or subclasses.
[0127] Antibodies of the present disclosure are generally isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. As used herein, “recombinant antibody” refers to an antibody that is generated using recombinant nucleic acid techniques in exogenous host cells, and recombinant antibodies can be isolated as well.
[0128] “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
[0129] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CHI, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
[0130] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites. The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) or complementary determining regions (CDRs), both in the light- chain and the heavy-chain variable domains, that confer antigen specificity. A “variable heavy domain” pairs with a “variable light domain” to form an antigen-binding domain (ABD) that specifically binds a target antigen. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs/HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs/HVRs in each chain are held together in close proximity by the FR regions and, with the CDRs/HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
[0131] The terms “hypervariable region,” “HVR,” “HV,” “complementary determining region,” and “CDR,” used interchangeably herein, refer to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs or CDRs; three in the VH (Hl, H2, H3; or VH CDR1, VH CDR2, VH CDR3), and three in the VL (LI, L2, L3; or VL CDR1, VL CDR2, VL CDR3).
[0132] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“ ”), based on the amino acid sequences of their constant domains.
[0133] Together, the CDRs of the VH and VL domains form an Fv region. In embodiments, a VH and a VL domain comprise the six CDRs of the ABD. In a “Fab” format, the variable heavy domain (VH; containing VH CDR1, VH CDR2, and VH CDR3) and the variable light domain (VL or VL; containing the VL CDR1, VL CDR2 and VL CDR3), comprise the set of 6 CDRs, with the C-terminus of the VH domain being attached to the N-terminus of the CHI domain of the heavy chain and the C-terminus of the VL domain being attached to the N-terminus of the constant light domain (and thus forming the light chain). In an “scFv” format, the VH and VL domains are covalently attached, generally through the use of a linker (e.g., an “scFv linker”), into a single polypeptide sequence, which can have the N- to C-terminus arrangement of VH-linker-VL or VL- linker-VH. In general, the C-terminus of the scFv domain is attached to the N-terminus of the hinge in the second monomer.
[0134] ‘Fab” or “Fab region,” as used herein, refers to a polypeptide that comprises VH, CHI , VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g., VH- CHI on one chain and VL-CL on the other). Fab can refer to this region in isolation, or this region in the context of an antibody of the disclosure. In embodiments, a Fab comprises an Fv region in addition to CHI CL domains.
[0135] Another part of the heavy chain is the hinge region. As used herein, “hinge,” “hinge region,” “antibody hinge region,” or “hinge domain” refers to the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CHl domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Thus, for IgG, the antibody hinge is herein defined to include positions 216 (E216 in IgGl) to 230 (p230 in IgGl), wherein the numbering is according to the EU index as in Kabat. In some cases, a “hinge fragment” is used, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain.
[0136] “Heavy chain constant region,” as used herein, refers to the CHl-hinge-CH2 -CH3 portion of an antibody or fragment thereof, excluding the variable heavy domain. In embodiments, the heavy chain constant region comprises amino acids 118-447 of human IgGl, in EU numbering. As used herein, “heavy chain constant region fragment” refers to a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region.
[0137] “Fv,” “Fv fragment,” or “Fv region,” as used herein, refers to a polypeptide that comprises VL and VH domains of an antibody binding domain. Fv regions can be formatted as both Fabs and scFvs, where the VL and VH domains are combined (e g., by way of a linker, as discussed herein) to form an scFv.
[0138] “Fc,” “Fc region,” or “Fc domain,” as used herein, refers to a polypeptide comprising CH2 -CH3 domains of an IgG molecule, and, in some cases, inclusive of the hinge. In EU numbering for human IgGl, the CH2 -CH3 domain comprises amino acids 231 to 447, and the hinge is 216 to 230. Thus, the definition of “Fc domain” includes both amino acids 231-447 (CH2 - CH3) and 216-447 (hinge-CH2 -CH3) of IgGl, or fragments thereof. An “Fc fragment” in this context can contain fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another Fc domain or Fc fragment as can be detected using standard methods, generally based on size (e g., non-denaturing chromatography, size exclusion chromatography, etc.). In embodiments, the disclosed AOCs comprise human Fc domains. In embodiments, the disclosed AOCs comprise Fc domains from human IgGl, IgG2, or IgG4.
[0139] A “variant Fc domain” contains amino acid modifications as compared to a parental Fc domain. Thus, a “variant human IgGl Fc domain” is one that contains amino acid modifications (generally amino acid substitutions, although in the case of ablation variants, amino acid deletions are included) as compared to the human IgGl Fc domain. In embodiments, variant Fc domains have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding parental human IgG Fc domain. In embodiments, the percent identity is calculated using the identity algorithms discussed below. In embodiments, the percent identity is calculated using the BLAST algorithm known in the art, using default parameters. In embodiments, variant Fc domains have from 1 to about 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid modifications as compared to the parental Fc domain. In embodiments, variant Fc domains retain the ability to form a dimer with Ir Fc domain as measured using known techniques as described herein, such as non-denaturing gel electrophoresis.
[0140] For all positions discussed in the present disclosure that relate to antibodies, unless otherwise noted, amino acid position numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof. See, SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al.; Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the contents of which are incorporated herein by reference. In embodiments of the present disclosure, amino acid position numbering is according to the IMGT system.
[0141] The terms “full length antibody,” “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region. [0142] An “antibody fragment” comprises a portion of an intact antibody, preferably comprising the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[0143] A “naked antibody” for the purposes herein is an antibody that is not conjugated to a payload, e.g., an oligonucleotide, cytotoxic moiety, or radiolabel.
[0144] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that can be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this disclosure. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
[0145] Antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit one or more of the desired biological activities (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or capability, while the constant regions are homologous to the sequences of antibodies derived from another species of mammals (e.g., human) to avoid eliciting an immune response In that species. Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
[0146] ‘Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In embodiments, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR/HVR of the recipient are replaced by residues from a CDR/HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593- 596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539 or 5,639,641, incorporated herein by reference in their entireties.
[0147] As used herein, the term “human antibody” refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
[0148] A “species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “binds specifically” to a human antigen (e.g., has a binding affinity (Kd) value of no more than about 1 X 10-7M, preferably no more than about U I0 x M and preferably no more than about 1 x 10 9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
[0149] The expression “linear antibodies” refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.
[0150] “Modification,” as used herein, refers to an amino acid substitution, insertion, deletion, and/or any other mutation in a polypeptide sequence. [0151] ‘Variant protein,” or “protein variant,” or “variant,” as used herein refers to a protein that differs from that of a parent protein by virtue of at least one amino acid modification. The protein variant has at least one amino acid modification compared to the parent protein, yet not so many that the variant protein will not align with the parental protein using an alignment program such as that described below. In general, variant proteins (such as variant Fc domains, etc., described herein, are generally at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% identical to the parent polypeptide, using any alignment program known in the art, such as BLAST.
[0152] Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as that of Smith, T.F. & Waterman, M.S. (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S.B. & Wunsch, CD. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol.48:443 [homology alignment algorithm], Pearson, W.R. & Lipman, D.J. (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [search for similarity method]; or Altschul, S.F. et al, (1990) “Basic Local Alignment Search Tool,” J. Mol. Biol. 215:403-10 , the “BLAST” algorithm, see the webpage located at URL blast.ncbi.nlm.nih.gov/Blast.cgi. When using any of the aforementioned algorithms, the default parameters (for Window length, gap penalty, etc.) are used. Unless specifically stated otherwise, sequence identity is determined using the BLAST algorithm, using default parameters.
[0153] In embodiments, a parent polypeptide, for example an Fc parent polypeptide, is a human wild type sequence, such as the heavy constant domain or Fc region from IgGl, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as “parent polypeptides.” In embodiments, antibody sequences described herein have at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with a parent polypeptide sequence. Accordingly, “antibody variant” or “variant antibody” as used herein refers to an antibody that differs from a parent antibody by virtue of at least one amino acid modification; “IgG variant” or “variant IgG” as used herein refers to an IgG that differs from a parent IgG (e.g., from a human IgG sequence) by virtue of at least one amino acid modification; “immunoglobulin variant” or “variant immunoglobulin” as used herein refers to an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification; and “Fc variant” or “variant Fc” as used herein refers to an Fc that differs from a parent Fc, e.g., an Fc domain of human IgGl, IgG2, IgG3, or IgG4, by virtue of at least one amino acid modification.
[0154] “IgG subclass modification” or “isotype modification,” as used herein, refers to amino acid modifications that convert one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, because IgGl comprises a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an IgG subclass modification.
[0155] ‘Non-naturally occurring modification” as used herein is meant an amino acid modification that is not isotypic. For example, because none of the human IgGs comprise a serine at position 434, the substitution 434S in IgGl, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification.
[0156] As used herein, “oligonucleotide” or “polynucleotide,” used interchangeably, refers to a linear polymer of natural or modified nucleoside monomers linked by phosphodiester bonds or analogs thereof. The term “oligonucleotide” usually refers to a shorter polymer, e.g., comprising from about 3 to about 100 monomers, and the term “polynucleotide” usually refers to longer polymers, e.g., comprising from about 100 monomers to many thousands of monomers, e.g., 10,000 monomers, or more. Oligonucleotides and polynucleotides can be natural or synthetic. Oligonucleotides and polynucleotides can include deoxyribonucleosides, ribonucleosides, and/or non-natural analogs thereof. In embodiments, oligonucleotides or polynucleotides are capable of specifically binding to a target genome by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. As used herein, “functional nucleic acid” refers to a nucleic acid having biological functions in vivo or in cells, such as enzymatic functions, catalytic functions, or biologically inhibiting or enhancing functions (e.g., inhibition or enhancement of transcription or translation). In embodiments, examples of functional nucleic acids include, but are not limited to, siRNA, ASO, shRNA, miRNA (including pri-miRNA and pre-miRNA), nucleic acid aptamers (including RNA aptamers and DNA aptamers), ribozymes (including deoxyribozymes), riboswitches, U1 adaptors, molecular beacons, and transcriptional factor- binding regions.
[0157] In certain instances, the term “oligonucleotide” is used in reference to an “antisense oligonucleotide.” For “antisense oligonucleotides,” each subunit consists of: (i) a ribose sugar or a derivative thereof; and (ii) a nucleobase bound thereto, such that the order of the base-pairing moieties forms a base sequence that is complementary to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence with the proviso that either the subunit, the intersubunit linkage, or both are not naturally occurring. In certain embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In other embodiments, the antisense oligonucleotide is a 2'-O-methyl phosphorothioate (2’OMe-PS). In other embodiments, the antisense oligonucleotide is a 2’-fluoro phosphorothioate (2’F-PS). In other embodiments, the antisense oligomer of the disclosure is a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or a bridged nucleic acid (BNA) such as 2'-O,4'-C-ethylene-bridged nucleic acid (ENA).
[0158] Morpholinos as described herein include all stereoisomers and tautomers of the foregoing general structure. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063; 5,506,337; 8,076,476; and 8,299,206; all of which are incorporated herein by reference.
[0159] The terms “complementary” and “complementarity” refer to two or more oligomers (i.e., each comprising a nucleobase sequence) that are related with one another by Watson-Crick base-pairing rules. For example, the nucleobase sequence “T-G-A (5'— >3'),” is complementary to the nucleobase sequence “A-C-T (3'— >5').” Complementarity may be “partial,” in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules. For example, in some embodiments, complementarity between a given nucleobase sequence and the other nucleobase sequence may be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Or, there may be “complete” or “perfect” (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example. The degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.
[0160] The terms “nucleobase” (Nu), “base pairing moiety” or “base” are used interchangeably to refer to a purine or pyrimidine base found in naturally occurring, or “native” DNA or RNA (e.g., uracil, thymine, adenine, cytosine, and guanine), as well as analogs of these naturally occurring purines and pyrimidines. These analogs may confer improved properties, such as binding affinity, to the oligomer. Exemplary analogs include hypoxanthine (the base component of inosine); 2,6-diaminopurine; 5-methyl cytosine; C5-propynyl-modified pyrimidines; 10-(9- (aminoethoxy)phenoxazinyl) (G-clamp) and the like.
[0161] The terms “mismatch” or “mismatches” refer to one or more nucleobases (whether contiguous or separate) in an oligomer nucleobase sequence that are not matched to a target pre- mRNA according to base pairing rules. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target pre-mRNA. Variations at any location within the oligomer are included. In certain embodiments, antisense oligomer conjugates of the disclosure include variations in nucleobase sequence near the term variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 subunits of the 5' and/or 3' terminus.
[0162] As used herein, a “3E10 antibody” refers to an antibody with a set of heavy chain CDRs (VH CDR1, VH CDR2, and VH CDR3), identified according to the Kabat system, comprising amino acid sequences that vary from SEQ ID NOS: 58, 59, and 60 by no more than two amino acids each, respectively, a set of light chain CDRs (VL CDR1, VL CDR2, and VL CRD3) comprising amino acid sequences that vary from SEQ ID NOS: 61, 62, and 63 by no more than two amino acids each, respectively, that binds nucleic acids and is cell-penetrating at least when bound to a nucleic acid, as well as antigen-binding fragments thereof. As described herein, the 3E10 antigen is a polynucleotide. Although generally referred to herein as “3E10” or “3E10 antibodies,” it will be appreciated that fragments, variants, and binding proteins, including antigen- binding fragments and fusion proteins, such as scFv, di-scFv, tr-scFv, and other single chain variable fragments, and other cell-penetrating, nucleic acid transporting molecules disclosed herein, are encompassed by the phrase and are also expressly provided for use in compositions, conjugates, and methods disclosed herein. Thus, the antibodies and other binding proteins are also referred to herein as cell-penetrating.
[0163] As used herein, the term “cell-penetrating” refers to an antibody or antigen binding fragment thereof that can penetrate a cell, e.g., a mammalian cell, without the aid of an exogeneous transport vehicle, such as a liposome, or a conjugated cell-penetrating peptide. With respect to 3E10 antibodies and antigen binding fragments thereof, the cell-penetrating antibody or antigen binding fragment thereof can penetrate a cell expressing an ENT2 receptor on its cell surface in the presence of nucleic acids, e.g., non-covalently bound and/or conjugated to the 3E10 antibody or antigen binding fragment thereof, resulting in internalization of the 3E10 antibodies and antigen binding fragments thereof. In some embodiments, the cell-penetrating 3E10 antibody or antigen binding fragment thereof is conjugated to a functional molecule, e.g., a chemical agent, polynucleotide, or polypeptide. Although the cell-penetrating molecules are generally referred to herein as “cell-penetrating antibodies,” it will be appreciated that fragments, including antigen-binding fragments, variants, binding proteins and fusion proteins such as scFv, di- scFv, tri-scFv, and other single chain variable fragments, and other cell-penetrating molecules disclosed herein are also expressly provided foruse in compositions, conjugates, and methods disclosed herein. Autoantibodies against double-stranded deoxyribonucleic acid (dsDNA) are frequently identified in the serum of patients with systemic lupus erythematosus (SLE) and are often implicated in disease pathogenesis. Therefore, in embodiments, cell-penetrating antibodies (e.g., cell-penetrating anti-DNA antibodies) can be derived or isolated from patients with SLE or animal models of SLE.
[0164] As used herein, “antibody-oligonucleotide conjugate” or “AOC” refers to an antibody or antigen-binding fragment thereof that is covalently linked or conjugated to a biologically active molecule, for example an oligonucleotide or anti-tumor oligonucleotide, for example, an siRNA molecule, an antisense oligonucleotide.
[0165] As used herein, a “linker” is any chemical moiety that is capable of linking or connecting a molecule, including an oligonucleotide, to a cell-binding agent such as an antibody, such as a 3E10 antibody or a fragment thereof, in a stable, covalent manner. In embodiments, a “linker” is any chemical moiety that is capable of linking or connecting a compound such as an oligonucleotide, a polynucleotide, a DNA damage-inducing agent, a DNA repair inhibitor, an immune modulatory molecule, an alkylating agent, a microtubule inhibitor, an immune checkpoint inhibitor, an angiogenesis inhibitor, an adoptive cell therapy, or a topoisomerase inhibitor, to a cell-binding agent such as a 3E10 antibody or a fragment thereof, in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, at conditions under which the compound and/or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.
[0166] As used herein, the term “subject” means any individual who is the target of administration. The subject can be any animal (e.g., a mammal. Thus), including, but not limited to, humans, and non-human animals (including, but not limited to, non-human primates, dogs, cats, rodents, horses, cows, pigs, mice, rats, hamsters, rabbits, and the like (e.g., which is to be the recipient of a particular treatment). In embodiments, the subject is a human. In embodiments, methods of the disclosure are useful in treatment a human subject. In embodiments, the human may be referred to as a patient. In embodiments, the human is a female. In embodiments, the human is a male. In embodiments, the human has an age in a range of from about 1 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
[0167] The terms “subject” and “patient” as used herein include any animal that exhibits a symptom, or is at risk for exhibiting a symptom, which can be treated with an antisense oligomer conjugate of the disclosure, such as a subject (or patient) that has or is at risk for having DMD or BMD, or any of the symptoms associated with these conditions (e.g., muscle fiber loss). Also included are methods of producing dystrophin in a subject (or patient) having a mutation of the dystrophin gene that is amenable to exon 23 skipping.
[0168] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, brain cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphomas, Hodgkin lymphoma, Non-Hodgkin lymphoma, cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, leukemia, neuroblastoma, Wilms tumor, bone cancer, brain stem tumor, childhood diffuse intrinsic pontine glioma (DIPG), retinoblastoma, rhabdoid tumor, sarcoma, spinal cord tumor, endocrine cancer, esophageal cancer, gastric cancer, hepatobiliary cancer, myeloma, renal cancer, thyroid cancer, uterine cancer, carcinoma, blastoma, papilloma, adenoma, an astrocytic tumor, an oligodendroglial tumor, an oligoastrocytic tumor, an ependymal tumor, a choroid plexus tumor, a neuronal or mixed neuronal-glial tumor, tumor of the pineal region, embryonal tumor, or an otherwise uncategorized neuroepithelial tumors.
[0169] “Tumor” and “neoplasm” refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous), including pre- cancerous lesions.
[0170] The terms “cancer cell,” “tumor cell,” and grammatical equivalents thereof refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non- tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
[0171] As used herein, the term “pharmaceutically effective amount” means that the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, as well as the route of administration and the pharmacokinetics of the agent being administered.
[0172] As used herein, the term “carrier” or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined. In embodiments, the carrier or excipient is selected to minimize degradation of the active ingredient or to minimize adverse side effects in the subject, as would be well known to one of skill in the art.
[0173] As used herein, the term “treat” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
[0174] As used herein “antibody-oligonucleotide conjugate” or “AOC” refers to an antibody or antigen-binding fragment thereof that is conjugated via a linker to a therapeutic oligonucleotide, for example, an oligonucleotide, an siRNA, or an antisense oligonucleotide (ASO), which may be delivered to specific cells or tissues otherwise not targetable by oligonucleotide delivery. The conjugation of an oligonucleotide with an antibody or antigen- binding fragment thereof may also improve the pharmacokinetic properties of therapeutic oligonucleotides, expanding application of this therapeutic modality.
[0175] A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce a desired physiological effect or amount capable of achieving a desired result, particularly for treating or preventing the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
[0176] The terms “effective amount” and “therapeutically effective amount” are used interchangeably herein and refer to an amount of therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect. For an antisense oligomer, this effect is typically brought about by inhibiting translation or natural splice-processing of a selected target sequence, or producing a clinically meaningful amount of dystrophin.
[0177] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures, tissue samples, tissue homogenates or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population) or the maximum tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In embodiments, compositions, conjugates, and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays or measurements. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
[0178] In embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
[0179] By “enhance” or “enhancing,” or “increase” or “increasing,” or “stimulate” or “stimulating,” refers generally to the ability of one or more antisense oligomer conjugates or pharmaceutical compositions to produce or cause a greater physiological response (i.e., downstream effects) in a cell or a subject, as compared to the response caused by either no antisense oligomer conjugate or a control compound. A greater physiological response may include increased expression of a functional form of a dystrophin protein, or increased dystrophin- related biological activity in muscle tissue, among other responses apparent from the understanding in the art and the description herein. Increased muscle function can also be measured, including increases or improvements in muscle function by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of muscle fibers that express a functional dystrophin can also be measured, including increased dystrophin expression in about 1%, 2%, 5%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of muscle fibers. For instance, it has been shown that around 40% of muscle function improvement can occur if 25-30% of fibers express dystrophin (see, e.g., DelloRusso et al, Proc Natl Acad Sci USA 99: 12979-12984, 2002). An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more times (e g., 500, 1000 times, including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) the amount produced by no antisense oligomer conjugate (the absence of an agent) or a control compound.
[0180] As used herein, the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.
[0181] A “functional” dystrophin protein refers generally to a dystrophin protein having sufficient biological activity to reduce the progressive degradation of muscle tissue that is otherwise characteristic of muscular dystrophy, typically as compared to the altered or “defective” form of dystrophin protein that is present in certain subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD). In certain embodiments, a functional dystrophin protein may have about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (including all integers in between) of the in vitro or in vivo biological activity of wild-type dystrophin, as measured according to routine techniques in the art. As one example, dystrophin-related activity in muscle cultures in vitro can be measured according to myotube size, myofibril organization (or disorganization), contractile activity, and spontaneous clustering of acetylcholine receptors (see, e.g., Brown et al., Journal of Cell Science. 112:209-216, 1999). Animal models are also valuable resources for studying the pathogenesis of disease, and provide a means to test dystrophin-related activity. Two of the most widely used animal models for DMD research are the mdx mouse and the golden retriever muscular dystrophy (GRMD) dog, both of which are dystrophin negative (see, e.g., Collins & Morgan, Int J Exp Pathol 84: 165-172, 2003). These and other animal models can be used to measure the functional activity of various dystrophin proteins. Included are truncated forms of dystrophin, such as those forms that are produced following the administration of certain of the exon-skipping antisense oligonucleotides of the present disclosure.
[0182]
[0183] The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
[0184] The phrase “targeting sequence” refers to a sequence of nucleobases of an oligomer that is complementary to a sequence of nucleotides in a target pre-mRNA. For example, in some embodiments of the disclosure, the sequence of nucleotides in the target pre-mRNA is an exon 23 annealing site in the dystrophin pre-mRNA.
[0185] As used herein, the term “treatment” of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the subject or cell. Treatment includes, but is not limited to, administration of an oligomer or a pharmaceutical composition thereof, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Treatment includes any desirable effect on the symptoms or pathology of a disease or condition associated with the dystrophin protein, as in certain forms of muscular dystrophy, and may include, for example, minimal changes or improvements in one or more measurable markers of the disease or condition being treated. Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.
Ill, Antibodv-Oligonucleotide Conjugates (AOCs)
[0186] The present disclosure is directed, in part, to antibody-oligonucleotide conjugates (AOCs) comprising a cell-penetrating antibody, e.g., a 3E10 antibody or antigen-binding fragment thereof, conjugated via a linker to an oligonucleotide, e.g., a therapeutic oligonucleotide. In embodiments, an AOC described herein has the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, and P is an oligonucleotide moiety as described herein.
[0187] In some aspects, the present disclosure relates to the use of 3E10 antibodies, and derivatives thereof, for delivering antisense oligonucleotides amendable for exon skipping in tissues of a subject, including but not limited to skeletal muscle tissues for treatment of genetic skeletal muscle disorders. As is discussed below, the term antibody is used generally. Antibodies that find use in the present disclosure take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments, and mimetics, described herein in various embodiments.
[0188] In some aspects, the antibody is conjugated to the biologically active molecule via a linker. In some aspects, the antibody is a 3E10 antibody or antigen-binding fragment thereof, as described herein. In some aspects, the antibody is a humanized 3E10 antibody or antigen-binding fragment thereof, as described herein. Any variety of agents can be transported via conjugation to the 3E10 antibody or antigen-binding fragment thereof, or humanized 3E10 antibody or antigen- binding fragment thereof, herein, such as inorganic and organic molecules, pharmaceutical agents, drugs, peptides, proteins, genetic material, and the like. In some aspects, the antibody- oligonucleotide conjugate (AOC) comprises an oligonucleotide. A . A ntigen-Binding Domains (A BDs)
[0189] As used herein, the term “antigen-binding domain” or “ABD” refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope of an antigen. Thus, in embodiments, an ABD comprises a hypervariable region, optionally a VH and/or VL domain of an antibody, optionally at least a VH domain. In embodiments, an ABD comprises at least one complementarity determining region (CDR) of an antibody. In embodiments, an ABD comprises at least two CDRs of an antibody. In embodiments, an ABD comprises at least three CDRs of an antibody. In embodiments, an ABD comprises at least four CDRs of an antibody. In embodiments, an ABD comprises at least five CDRs of an antibody. In embodiments, an ABD comprises six CDRs of an antibody.
1. 3E10 Antibodies
[0190] In embodiments, the present disclosure relates to the use of 3E10 antibodies and antigen binding fragments thereof, e.g., for delivering therapeutic agents (e.g., oligonucleotides) into a cell within a subject. Although generally referred to herein as “3E10,” “3E10 antibodies,” and the like, it will be appreciated that disclosure herein referring to such antibodies also encompass antigen-binding fragments thereof, e.g., scFv, di-scFv, tr-scFv, regardless of whether it is specifically recited in each instance. Thus, when describing a feature of a 3E10 antibody for use in the various compositions, conjugates, fusion proteins, and methods disclosed herein, that same feature is implicitly disclosed with respect to 3E10 antigen-binding fragments as well.
[0191] In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 3, 4, and 5. In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 22, 23, and 24 and VH CDRs of SEQ ID NOs: 15, 17, and 18. In embodiments, a 3E10 antibody comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 16, 4, and 5. Other examples of 3E10 VL and VH CDR sequences are shown in Figures 1-4.
[0192] 3E10 is known to interact with the ENT2 nucleoside transporter expressed on various cell types, including muscle cells and cancer cells. In fact, ENT2 is overexpressed in most, if not all cancers. Accordingly, an AOC provided herein can widely target cancers based on cell surface expression of ENT2 on cancer cells. Advantageously, an AOC described herein can target ENT2 and extracellular DN A simultaneously. Importantly, 3E10 has been shown to preferentially localize into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. Targeting of the 3E10 antibody to extracellular DNA is described in, for example in Weisbart, Sci Reports, 2015, which is herein incorporated by reference. By targeting ENT2 as well as extracellular DNA, an 3E10 AOC described herein presents a platform to target a variety of cancers and deliver therapeutic oligonucleotides to target and kill cancer cells.
[0193] In some aspects of the present disclosure, the antibody or antigen-binding fragment thereof is a murine, chimeric, humanized, or human antibody or antigen-binding fragment thereof.
[0194] In some aspects, an AOC of the present disclosure take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments, and mimetics, described herein in various embodimentspenetrates into cells and nuclei in an ENT2- dependent manner.
[0195] In embodiments, a second polynucleotide is non-covalently bound to an AOC of the present disclosure, to help facilitate cellular internalization of the AOC. That is, in some embodiments, polynucleotides conjugated to the antibody (cargo polynucleotides) do not interact with the nucleic acid-binding paratope of the antibody, and a second polynucleotide (e.g., carrier nucleic acid) is non-covalently complexed with the paratope to help facilitate internalization. In embodiments, the second polynucleotide is precomplexed with the AOC prior to administering the AOC to a subject. In embodiments, the second polynucleotide is an extracellular polynucleotide that is bound by the AOC at a site of interest in vivo, for example, at a site of tumor ischemia and/or necrosis. In embodiments, the second polynucleotide is DNA. In embodiments, the second polynucleotide is RNA.
[0196] In embodiments, an AOC disclosed herein comprises a VH and VL domain of a 3E10 antibody. In embodiments, the AOC comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a 3E10 antibody.
[0197] In some aspects, the present disclosure provides an antibody-oligonucleotide conjugate having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, and P is an oligonucleotide as described herein, wherein the linker L links A to P. In embodiments, L is a cleavable linker and P is an ASO that mediates exon skipping. Tn some aspects of the present disclosure, the amino acid residue corresponding with D31 of the heavy chain CDR1 of the 3E10 antibody or antigen-binding fragment thereof is substituted with N. It is known in the art that mutation of aspartic acid at residue 31 of VH CDR1 to asparagine increases the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N). In embodiments, additional 3E10 antibody variants include mutation of aspartic acid at residue 31 of VH CDR1 to arginine (3E10-D31R), which modeling indicates expands cationic charge, or lysine (3E10-D31K) which modeling indicates changes charge orientation. Thus, in some aspects, the 3E10 antibody or antigen-binding fragment thereof includes a D31R or D3 IK substitution. In embodiments, additional 3E10 antibody variants include R96N, and/or S30D, alone or in combination with D31N, D31R, or D31K. All of the sequences disclosed herein having the residue corresponding to 3E10 D31 or N31, are expressly disclosed with a D31R or D3 IK or N31R or N3 IK substitution.
[0198] In embodiments, the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is an oligonucleotide as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, wherein the linker L links A to (P); wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of XI YGMX2, where XI is D, E, N, Q, R, or K and X2 is K, R, or H (SEQ ID NO:58). In embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of X1ASX2X3VSTSSYSYX4X5, where XI is K, R, or H, X2 is K, R, or H, X3 is T or S, X4 is M or L, and X5 is K, R, H, or A (SEQ ID NO:61), (b) a VL CDR2 comprising the amino acid sequence of YASYLX1S, where XI is D, E, N, or Q (SEQ ID NO:62), and (c) a VL CDR3 comprising the amino acid sequence of QX1SX2X3FPWT, where XI is K, R, or H, X2 is K, R, or H, and X3 is D or E (SEQ ID NO:63), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of X1YGMX2, where XI is D, E, N, Q, R, or K and X2 is K, R, or H (SEQ ID NO: 58), (e) a VH CDR2 comprising the amino acid sequence of YISSX1SSTIYYAX2X3VX4G, where XI is G or S, X2 is D or E, X3 is T or S, and X4 is K, R, or H (SEQ ID NO:59), and (f) a VH CDR3 comprising the amino acid sequence of X1GLLLX2Y, where XI is K, R, or H, and X2 is D or E (SEQ ID NO:60). [0199] In embodiments, the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, wherein the linker L links A to P; wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15). In embodiments, the antibody or antigen- binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:9), (b) a VL CDR2 comprising the amino acid sequence of YASYLES (SEQ ID NO: 10), and (c) a VL CDR3 comprising the amino acid sequence of QHSREFPWT (SEQ ID NO: 11), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15), (e) a VH CDR2 comprising the amino acid sequence of YISSGSSTIYYADTVKG (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of RGLLLDY (SEQ ID NO: 5). In embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence that is identical to SEQ ID NO:21. In embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is identical to SEQ ID NO: 14. In embodiments, the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence that is identical to SEQ ID NO:20. In embodiments, the antibody or antigen-binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is identical to SEQ ID NO: 13.
[0200] In embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:21. In embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 14. In embodiments, the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:20. In embodiments, the antibody or antigen-binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13.
[0201] In some aspects, the 3E10 antibody or antigen-binding fragment thereof can be transported into the cytoplasm and/or nucleus of the cells without the aid of a carrier or conjugate. For example, a monoclonal 3E10 antibody and active fragments thereof that are transported in vivo to the nucleus of mammalian cells without cytotoxic effect are disclosed in U.S. Patent Nos. 4,812,397 and 7,189,396 to Richard Weisbart, the disclosures of which are incorporated by reference herein, in their entireties.
[0202] Amino acid sequences of 3E10 monoclonal antibodies and antigen-binding fragments thereof are known in the art. Example sequences of 3E10 heavy and light chains are provided below herein.
[0203] A murine version of the 3E10 antibody is described in Zack, et al., Immunology and Cell Biology, 72:513-520 (1994), the disclosure of which is incorporated by reference herein, in its entirety.
[0204] Amino acid variants of the 3E10 antibody are also known in the art, for example, as described in Zack, et al., J. Immunol., 157(5):2082-8 (1996). For example, amino acid position 31, in CDR1 of the heavy chain variable region of 3E10, influences nucleic acid binding and the antibody’s ability to penetrate nuclei. Substitution of the ‘wild-type’ (e.g., relative to the original murine antibody) aspartic acid by asparagine (the ‘D3 IN’ mutation) improves nucleic acid binding and nuclei penetration of the antibody, relative to the ‘wild type’ murine antibody. See, for example, Zack, et al., Immunology and Cell Biology, 72:513-520 (1994); Weisbart, et al., J. Autoimmun ., 11, 539-546 (1998); and Weisbart, Int. J. Oncol., 25, 1867-1873 (2004) (which are incorporated by reference herein, in their entireties).
[0205] Sequences for 3E10 antibodies and antigen-binding fragments or variants thereof, with the D3 IN substitution, are disclosed herein. In some aspects, the 3E10 antibodies and antigen- binding fragments thereof disclosed herein include the D31N substitution. In some aspects, other amino acids are substituted at position 31 in the 3E10 antibodies and antigen-binding fragments thereof disclosed herein. For example, D31R, D31K, or D31R substitutions are incorporated in some aspects of the present disclosure.
[0206] Other 3E10 light chain sequences are known in the art. See, for example, Zack, et al., J. Immunol., 15;154(4):1987-94 (1995); GenBank: L16981.1 - Mouse Ig rearranged L-chain gene, partial cds; GenBank: AAA65681.1 - immunoglobulin light chain, partial [Mus musculus]).
[0207] Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. In embodiments, an antibody disclosed herein is an IgA, IgD, IgE, IgG, or IgM antibody, including any subtype or isotype thereof. In embodiments, an antibody disclosed herein is based on the IgG class, which has several. In embodiments, an antibody disclosed herein is based on one of the subclasses of IgG, including, but not limited to IgGl, IgG2, IgG3, and IgG4. In general, IgGl, IgG2 and IgG4 are used more frequently than IgG3. It should be noted that IgGl has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M), and in embodiments, antibodies disclosed herein are based on IgGl having D or E at position 356 and/or L or M at position 358.
[0208] The light chain generally comprises two domains, the variable light domain (containing the light chain CDRs and together with the variable heavy domains forming the Fv region), and a constant light chain region (often referred to as CL or CK). The heavy chain comprises a variable heavy domain and a constant domain, which includes a CHI -optional hinge- Fc domain comprising a CH2 -CH3.
[0209] The hypervariable region of an antibody generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g.., residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs useful for the compositions, conjugates, and methods described herein are described below.
[0210] As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the VH CDRs (e.g. VH CDR1, VH CDR2., and VH CDR3) and the disclosure of each variable light region is a disclosure of the VL CDRs (e.g. VL CDR1, VL CDR2, and vlCDR3). A useful comparison of CDR numbering is described in Lafranc et al., Dev. Comp. Immunol. 27(1): 55-77 (2003)).
[0211] Throughout the present disclosure, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)). In some aspects, the present specification uses the IMGT system to define the complementarity determining regions (CDRs) provided herein.
[0212] The present disclosure provides a large number of different CDR sets. In this case, a “full CDR set” comprises the three variable light CDRs, e.g., a VL CDR1, VL CDR2, and VL CDR3, and the three variable heavy CDRs, e.g., a VH CDR1, VH CDR2, and VH CDR3. These can be part of a larger variable light or variable heavy domain, respectfully. In addition, as more fully outlined herein, the variable heavy and variable light domains can be on separate polypeptide chains, when a heavy and light chain is used (for example when Fabs are used), or on a single polypeptide chain in the case of scFv sequences.
[0213] The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as nucleic acids, amino acids, or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. The antibodies described herein bind to nucleic acid epitopes in a partially sequence-independent manner. That is, while the antibodies described herein bind to some polynucleotide structures and sequences with greater affinity than other nucleic acid structures and sequences, they have some general affinity for polynucleotides.
[0214] The “Fc domain” of the heavy chain includes the -CH2 -CH3 domain, and optionally a hinge domain (-H-CH2 -CH3). For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cy2 and Cy3) and the lower hinge region between CHI (Cyl) and CH2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. Accordingly, “CH” domains in the context of IgG are as follows: “CHI” refers to positions 118-215 according to the EU index as in Kabat. “Hinge” refers to positions 216-230 according to the EU index as in Kabat. “CH2” refers to positions 231-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat. Thus, the “Fc domain” includes the -CH2 -CH3 domain, and optionally a hinge domain (hinge-CH2 -CH3). In the embodiments herein, when a scFv is attached to an Fc domain, it is generally the C-terminus of the scFv construct that is attached to all or part of the hinge of the Fc domain; for example, it is generally attached to the sequence EPKS which is the beginning of the hinge. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcyR receptors or to the FcRn receptor, and to enable heterodimer formation and purification, as outlined herein.
[0215] Another part of the heavy chain is the hinge region. By “hinge” or “hinge region” or “antibody hinge region” or “hinge domain” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. As noted above herein, the present disclosure refers to different antibody domains of a 3E10 antibody or antigen- binding fragment thereof. Structurally, the IgG CHI domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Thus for IgG the antibody hinge is herein defined to include positions 216 (E216 in IgGl) to 230 (p230 in IgGl), wherein the numbering is according to the EU index as in Kabat. In some cases, a “hinge fragment” is used, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain.
[0216] A scFv comprises a variable heavy chain, an scFv linker, and a variable light domain. In most of the constructs and sequences outlined herein, the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable light chain (N-vh-linker-vl-C) although that can be switched (N- vl-linker-vh-C).
[0217] Thus, the present disclosure relates to different antibody domains. These domains include, but are not limited to, the Fc domain, the CHI domain, the CH2 domain, the CH3 domain, the hinge domain, the heavy constant domain (CHl-hinge-Fc domain or CHl-hinge-CH2 -CH3), the variable heavy (VH) domain, the variable light (VL) domain, the light constant domain, Fab domains and scFv domains.
2. Humanized Antibodies
[0218] In certain embodiments, the antibodies of the disclosure comprise a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene. For example, in some embodiments, such an antibody comprises or consists of a murine, chimeric, humanized, or human antibody or antigen-binding fragment thereof comprising heavy or light chain variable regions that are "“the product of'” or "“derived from"” a particular germline sequence, e.g., that of the 3E10 antibody. A human antibody that is "“the product of1” or "“derived from"” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody (using the methods outlined herein). A human antibody that is “the product of’ or “derived from” a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation. However, a humanized antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as being derived from human sequences when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In some embodiments, a humanized antibody is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a humanized antibody derived from a particular human germline sequence will display no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some embodiments, the humanized antibody has no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
[0219] In some embodiments, a parental antibody is affinity matured. Methods for affinity maturation are known in the art. In some embodiments, structure-based methods are employed for humanization and affinity maturation, for example, as described in U.S Patent Publication No. 2006/0008883, which is incorporated herein by reference. Selection based methods are also known for humanization and/or affinity maturation of antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294: 151-162; Baca et al., 1997, J. Biol. Chem. 272(16): 10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are incorporated herein by reference. Yes other known humanization methods include grafting of only parts of the CDRs, including but not limited to methods described in U.S Patent Patent Publication No. 2001/0035606; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all of which are incorporated herein by reference.
3. Fc variants
[0220] In embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
[0221] In embodiments, an Fc region variant possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays known in the art can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006)).
[0222] In embodiments, an antibody provided herein can have reduced effector function and thus can comprise a substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
[0223] In embodiments, an Fc region variant provided herein can have improved or diminished binding to FcRs. See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001), the disclosure of which are incorporated herein by reference, in their entireties.
[0224] In embodiments, an Fc region variant provided herein comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
[0225] In embodiments, an Fc region variant provided herein comprises alterations that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0226] In embodiments, an Fc region variant provided herein comprises alterations that result in increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), e.g., as described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more ofFc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
[0227] In embodiments, an Fc region variant provided herein comprises “knob-in-hole” or “skew” variants, which refer to amino acid engineering that creates stearic influences to favor heterodimeric formation and disfavor homodimeric formation, as described in USSN 61/596,846, Ridgway et al, Protein Engineering 9(7):617 (1996); Atwell et al, J. Mol. Biol. 1997 270:26; US Patent No. 8,216,805, all of which are hereby incorporated by reference in their entirety.
[0228] In embodiments, an Fc region variant provided herein comprises alterations described in Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351.
4. Antibody Fragments
[0229] In embodiments, the antibody portion of an AOC described herein comprises an antigen-binding fragment of a 3E10 antibody. In embodiments, the antigen-binding fragment retains the desired biological activity of a 3E10 antibody. In embodiments, the antigen-binding fragment retains at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the desired biological activity of a 3E10 antibody. In embodiments, the antigen-binding fragment retains the ability of the antibody to bind to its target antigen, e.g., a nucleic acid, e.g., DNA. In embodiments, the antigen-binding fragment retains the ability of the antibody to bind to a cellular receptor, e.g., ENT2. In embodiments, the antigen- binding fragment retains the ability of the antibody to be internalized by a target cell.
[0230] In embodiments, the 3E10 antibody or antigen binding fragment thereof comprises a single-chain fragment variable (scFv), a tandem double scFv, an (scFv)2, a minibody, a VHH, an scFv-Fc, a CrossMab, a dual variable domain immunoglobulin (DVD-Ig), a single-chain tandem fragment variable (scTaFv), a diabody, a tandem diabody (TandAb), a Fabsc, a modular IgG-scFv, a Fab, or an F(ab’)2.
[0231] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a CrossMab. In the CrossMab format, complementary mutations are introduced in the heavy chain constant region of each arm to generate so-called “holes and knobs,” resulting in preferred association between different arms, forming a heterodimer, rather than a homodimer of two of the same arms. The exact residues that are mutated in the heavy chain constant region of a CrossMab bispecific antibody to form “holes” and “knobs” can vary depending on the specific design and optimization goals of the antibody. For more information on CrossMab antibodies see, for example, Huang, J., et al., Journal of Biological Chemistry, 294(50): 19001-10 (2019), the disclosure of which is incorporated herein by reference in its entirety.
[0232] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a divalent, dual-variable domain immunoglobulin (DVD-Ig) of a 3E10 antibody or antigen binding fragment thereof. In the DVD-Ig format, each arm of the antibody contains two VH/VL pairs. In some embodiments, one of the VH/VL pairs comprises 3E10 VH and VL CDRs. For more information on DVD-Ig antibodies see, for example, Polson AG, et al., Journal of immunotherapy. 29(3):241-50 (2006) and U.S. Patent No. 7,612,181, the disclosures of which are incorporated herein by reference in their entireties.
[0233] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a single-chain variable fragment (scFv). “Single chain Fv” or “scFv,” as used herein, refers to a VH domain covalently attached to a VL domain through a linker, e g., a scFv linker as discussed herein, to form a continuous protein chain. A scFv domain can be in either arrangement from N- to C-terminus (i.e., VH-linker-VL or VL-linker-VH). In the sequences depicted in the sequence listing and in the figures herein, the order of the VH and VL domain is indicated in the name, e.g., H.X L.Y means the N- to C-terminus arrangement is VH- linker-VL, and L.Y H.X means the N- to C-terminus arrangement is VL-linker-VH.
[0234] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a tandem double scFv. A tandem double scFv has two scFv domains linked in a linear fashion. In some embodiments, each scFv domain is derived from a different antibody and provides independent antigen-binding specificity. In some embodiments, one of the scFv domains comprises 3E10 VH and VL CDRs. For more information on tandem double scFvs see, for example, Bossen C, et al., MAbs, 4(2):200-08 (2012), the disclosure of which is incorporated herein by reference in its entirety. [0235] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a dimeric scFv antibody (scFv)2. A dimeric scFv antibody has two scFv domains linked in a dimeric arrangement. In some embodiments, each scFv domain is derived from a different antibody and provides independent antigen-binding specificity. In some embodiments, one of the scFv domains comprises 3E10 VH and VL CDRs. For more information on dimeric scFv antibodies see, for example, Llewellyn C, et al., Journal of immunological methods, 273(l-2):33-44 (2002), the disclosure of which is incorporated herein by reference in its entirety.
[0236] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a scFv-Fc. An “scFv-Fc,” as meant herein, is a polypeptide that consists of a heavy and a light chain variable region of an antibody joined by a linker, which is followed by an Fc polypeptide chain of an antibody, optionally the Fc region of a human IgG antibody, such as an IgGl, IgG2, IgG3, or IgG4 antibody.
[0237] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a single-chain tandem fragment variable (scTaFv) antibody. A single-chain tandem fragment variable (scTaFv) antibody is a type of bispecific antibody that consists of two variable fragment (VH and VL) domains linked in a tandem arrangement. In some embodiments, one of the variable fragment domains comprises 3E10 VH and VL CDRs. For more information on scTaFv antibodies see, for example, Schramm C, et al., MAbs 5(3):442-49 (2013), the disclosure of which is incorporated herein by reference in its entirety.
[0238] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a VHH, also referred to as a nanobody. As used herein, the term “VHH” refers to a variable domain of heavy chain of heavy-chain antibody. A VHH is a molecule that can recognize an antigen through a single domain and is the smallest unit among antibody molecules that have been found to date. In embodiments, a VHH can include one or more variable domains of heavy chain derived from a heavy-chain antibody, and the number of the variable domains of heavy chain included in the VHH is not limited.
[0239] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a diabody. As used herein, “diabody” refers to a divalent antibody comprising two polypeptide chains, wherein each polypeptide chain is too short for a pair to form between two domains on the same chain such that each domain is paired with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-48 and Poljak et al., 1994, Structure 2: 1121-23). If the two polypeptide chains of the diabody are identical, there will be two identical antigen-binding sites in the diabody resulting from their pairing. In embodiments, one of the antigen binding domains of the diabody comprises 3E10 VH and VL CDRs. For more information on diabodies see, for example, Hoogenboom HR, et al., Trends Biotechnol., 21 (12): 553-57 (2003), the disclosure of which is incorporated herein by reference in its entirety. Polypeptide chains of different sequences can be used to prepare diabodies with two different antigen-binding sites. Similarly, as used herein, “triabodies” and “tetrabodies” refer to antibodies that contain three and four polypeptide chains, respectively, and form three and four antigen-binding sites (which can be the same or different), respectively.
[0240] The term “minibody” is used to refer to an scFv-CH3 fusion protein that self- assembles into a bivalent dimer of 80 kDa (ScFv-CH3)2.
[0241] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a tandem diabody (TandAb). A tandem diabody has two antigen-binding domains (VH and VL) linked in a tandem arrangement by a flexible peptide linker. In some embodiments, one of the antigen binding domains comprises 3E10 VH and VL CDRs. For more information on diabodies see, for example, Sidelmann JG, et al., Mol Immunol., 45(9):2597-607 (2008), the disclosure of which is incorporated herein by reference in its entirety.
[0242] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a Fabsc. As used herein, a “Fabsc” format antibody molecule typically refers to a bispecific antibody molecule having a Fab fragment, which generally includes a hinge region, which is at the C-terminus of the Fab fragment linked to the N- terminus of a CH2 domain, of which the C-terminus is in turn linked to the N-terminus of a scFv fragment.
[0243] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises a scFab. A scFab, also known as a single-chain fragment antigen binding (Fab), is a type of antibody fragment that combines the variable heavy chain (VH) and variable light chain (VL) domains into a single polypeptide chain, linked by a peptide linker. The domain structure of a Fabsc includes the variable domains of both the heavy chain and light chain (VH and VL), and a peptide linker that connects the two domains. In addition to the variable domains, a Fabsc also includes the constant domains of the light chain (CL) and the hinge region of the heavy chain. In some embodiments, one of the antigen binding domains comprises 3E10 VH and VL CDRs. For more information on Fabscs see, for example, Kettner, C., et al., Frontiers in Immunology, 8(8):453 (2017), the disclosure of which is incorporated herein by reference in its entirety.
[0244] In embodiments, an antigen-binding fragment of a 3E10 antibody or antigen- binding fragment thereof comprises an IgG-scFv. An IgG-scFv is an antibody in which a scFv is fused to the light chain or heavy chain of an IgG. In some embodiments, the scFv comprises 3E10 VH and VL CDRs. In some embodiments, the IgG comprises 3E10 VH and V LCDRs. In some embodiments, the antibody is an F(ab’)2.
5. Bispecific Antibodies
[0245] In embodiments, 3E10 antibodies and antigen-binding fragments thereof can be modified to improve their therapeutic potential. For example, in embodiments, the cell-penetrating anti-DNA antibody is conjugated to another antibody specific for a second therapeutic target in the cytoplasm and/or nucleus of a target cell. For example, in embodiments, the cell-penetrating 3E10 antibody is a bispecific antibody having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds a second therapeutic target.
[0246] Bispecific antibodies and other binding proteins having a first heavy chain and a first light chain from 3E10 and a second heavy chain and a second light chain from a monoclonal antibody that specifically binds a second target are discussed in Weisbart, et al., Mol. Cancer Ther., 11(10):2169-73 (2012), and Weisbart, et al., Int. J. Oncology, 25: 1113-8 (2004), and U.S. Patent Application No. 2013/0266570, which are specifically incorporated by reference in their entireties. In embodiments, the second target is specific for a target cell-type, tissue, organ, etc. Thus the second heavy chain and second light chain can serve as a targeting moiety that targets the complex to the target cell-type, tissue, organ. In embodiments, the second heavy chain and second light chain target, hematopoietic stem cells, CD34+ cells, T cells or any another cell type of interest, e g., by targeting a receptor or ligand expressed on the cell type of interest. In embodiments, the second heavy chain and second light chain target the thymus, spleen, or cancer cells. [0247] Bispecific antibodies can be used to direct cytotoxic agents or drugs to cells which express a particular antigen. These antibodies possess two binding sites directed at two different antigens or two different epitopes on the same antigen. For example, in embodiments thebispecific can comprise one arm for ENT2 engagement and another arm for a second target. Bispecific antibody design can include a variety of antibody designs with multiple binding arms. Techniques for making bispecific antibodies are common in the art (Millstein et al., 1983, Nature 305:537- 539; Brennan et al., 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121 : 120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J. Immunol. 148: 1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Patent 5,731,168). Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60 (1991)). In embodiments the contemplated bispecific antibody disclosed herein can be conjugated as a bispecific antibody-payload conjugate.
[0248] Heteroconjugate antibodies are also within the scope of the present disclosure. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate. In embodiments the contemplated herteoconjugate antibody disclosed herein can be conjugated as a heteroconjugate antibody-payload conjugate.
6. 3E10 Sequences
[0249] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences corresponding to the parent 3E10 antibody.
[0250] Accordingly, in embodiments, a 3E10 antibody or antigen-binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of X1ASX2X3VSTSSYSYX4X5, where XI is K, R, or H, X2 is K, R, or H, X3 is T or S, X4 is M or L, and X5 is K, R, H, or A (SEQ ID NO:61), (b) a VL CDR2 comprising the amino acid sequence of YASYLX1S, where XI is D, E, N, or Q (SEQ ID NO:62), and (c) a VL CDR3 comprising the amino acid sequence of QX1 SX2X3FPWT, where XI is K, R, or H, X2 is K, R, or H, and X3 is D or E (SEQ ID NO:63), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of X1YGMX2, where XI is D, E, N, Q, R, or K and X2 is K, R, or H (SEQ ID NO: 58), (e) a VH CDR2 comprising the amino acid sequence of YISSX1SSTIYYAX2X3VX4G, where XI is G or S, X2 is D or E, X3 is T or S, and X4 is K, R, or H (SEQ ID NO: 59), and (f) a VH CDR3 comprising the amino acid sequence of X1GLLLX2Y, where XI is K, R, or H, and X2 is D or E (SEQ ID NO:60).
[0251] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDR1 (SEQ ID NO:3), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).
[0252] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences from a variant 3E10 antibody that includes a D31N amino acid substitution in the VH CDR1. Accordingly, in embodiments, the a 3E10 antibody or antigen- binding fragment thereof includes a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 D3 IN (SEQ ID NO:22), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 D31N (SEQ ID NO:23), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 D31N (SEQ ID NO:24), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH- CDR1 D31N (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence of 3E10-VH- CDR2 D31N (SEQ ID NO: 17), and a VH CDR3 comprising the amino acid sequence of 3E10- VH-CDR3 D3 IN (SEQ ID NO: 18).
[0253] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein refers to CDR sequences corresponding to the parent 3E10 antibody, optionally including a D3 IN amino acid substitution in the VH CDR1. Accordingly, in embodiments, a 3E10 antibody or antigen-binding fragment thereof includes a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO:4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO:5).
[0254] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences corresponding to the parent 3E10 antibody, with a known amino acid substitution in one or more CDR. Accordingly, in embodiments, a 3E10 antibody or antigen- binding fragment thereof described herein includes one or more amino acid substitution, relative to the CDR sequences of the parent 3E10 or 3E10-D31N variant, selected from a G to S substitution at position 5 of VH CDR2, a T to S substitution at position 14 of VH CDR2, an S to T substitution at position 5 of VL CDR1, an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2.
[0255] Accordingly, in embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.1 (SEQ ID NO:26) or 3E10-VH-CDR2.2 (SEQ ID NO:27). In embodiments, the 3E10 antibody or antigen- binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant.
[0256] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.1 (SEQ ID NO:28) or 3E10-VL- CDR1.2 (SEQ ID NO:29). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 according to the 3E10- D3 IN variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant.
[0257] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.1 (SEQ ID NO:30). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant.
[0258] While some of the amino acid substitutions described above are fairly conservative substitutions — e.g., an S to T substitution at position 5 of VL CDR1 — other substitutions are to amino acids that have vastly different properties — e.g., an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2. This suggests, without being bound by theory, that at least these positions within the 3E10 CDR framework are tolerant to other amino acid substitutions.
[0259] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH
CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.3 (SEQ ID NO:31). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant, e.g., as described herein.
[0260] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.3 (SEQ ID NO:32). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
2 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant, e g., as described herein.
[0261] In embodiments, a 3E10 antibody or antigen-binding fragment thereof, includes VL
CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.2 (SEQ ID NO:33). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody or relative to the 3E10- D3 IN variant, e g., as described herein.
[0262] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDRl.cl (SEQ ID NO:34), 3E10-VH- CDRl.c2 (SEQ ID NO:35), 3E10-VH-CDRl.c3 (SEQ ID NO:36), 3E10-VH-CDRl.c4 (SEQ ID NO:37), or 3E10-VH-CDRl.c5 (SEQ ID NO:38). In embodiments, the 3E10 antibody or antigen- binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 2 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 2 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0263] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.cl (SEQ ID NO:39), 3E10-VH- CDR2.c2 (SEQ ID NO:40), or 3E10-VH-CDR2.c3 (SEQ ID NO:41). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 -3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0264] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3.cl (SEQ ID NO:42), 3E10-VH- CDR3.c2 (SEQ ID NO:43), or 3E10-VH-CDR3.c3 (SEQ ID NO:44). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 -3, and VH CDRs 1 and 2 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0265] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.C1 (SEQ ID NO:45), 3E10-VL- CDR1.C2 (SEQ ID NO:46), 3E10-VL-CDRl.c3 (SEQ ID NO:47), 3E10-VL-CDRl.c4 (SEQ ID NO:48), 3E10-VL-CDR1.C5 (SEQ ID NO:49), or 3E10-VL-CDRl.c6 (SEQ ID NO:50). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
2 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-
3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0266] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.cl (SEQ ID NO:51). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1- 3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein. [0267] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3.cl (SEQ ID NO:52), 3E10-VL- CDR3.C2 (SEQ ID NO:53), 3E10-VL-CDR3.c3 (SEQ ID NO:54), 3E10-VL-CDR3.c4 (SEQ ID NO:55), 3E10-VL-CDR3.c5 (SEQ ID NO:56), or 3E10-VL-CDR3.c6 (SEQ ID NO:57). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
1 and 2, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1- 3 according to the 3E10- D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0268] It is also contemplated that a 3E10 antibody or antigen-binding fragment thereof, as described herein, includes any combination of the 3E10 CDR amino acid substitutions described above.
[0269] Accordingly, in embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO:58). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 2 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs
2 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0270] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO:59). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and
3 according to the 3E10-D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0271] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO:60). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 according to the 3E10-D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3, and VH CDRs 1 and 2 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0272] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO:61). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs
2 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-
3 according to the 3E10-D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e g., as described herein.
[0273] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO:62). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1 - 3 according to the 3E10-D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein.
[0274] In embodiments, a 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO:63). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1-3 according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1- 3 according to the 3E10-D31N variant. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2, and VH CDRs 1-3 having one or more amino acid substitutions relative to the CDRs of the parent 3E10 antibody, e.g., as described herein. [0275] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein includes a VL CDR 1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO:61), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO:62), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO:63), a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO:58), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO:59), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO:60).
[0276] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein refers to CDR sequences having no more than one amino acid substitution relative to the parent 3E10 antibody optionally including a D31N amino acid substitution in the VH CDR1. Accordingly, in embodiments, a 3E10 antibody or antigen-binding fragment thereof includes a VL CDR 1 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDR2 (SEQ ID NO:4), and a VH CDR3 comprising an amino acid sequence having no more than one amino acid substitution relative to 3E10-VH-CDR3 (SEQ ID NO: 5).
[0277] In embodiments, a 3E10 antibody or antigen-binding fragment thereof described herein refers to CDR sequences having no more than two amino acid substitution relative to the parent 3E10 antibody optionally including a D31N amino acid substitution in the VH CDR1. Accordingly, in embodiments, a 3E10 antibody or antigen-binding fragment thereof includes a VL CDR 1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR1 (SEQ ID NO:9), a VL CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable region (VH) CDR1 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR2 (SEQ ID NON), and a VH CDR3 comprising an amino acid sequence having no more than two amino acid substitutions relative to 3E10-VH-CDR3 (SEQ ID NO: 5).
[0278] Other variants of a 3E10 antibody or antigen-binding fragment thereof are also known in the art, as disclosed for example, in Zack, et al., J. Immunol., 157(5):2082-8 (1996). For example, amino acid position 31 of the heavy chain variable region of 3E10 has been determined to be influential in the ability of the antibody and fragments thereof to penetrate nuclei and bind to DNA. A D31N mutation in CDR1 penetrates nuclei and binds DNA with much greater efficiency than the original antibody (Zack, et al., Immunology and Cell Biology, 72:513-520 (1994), Weisbart, et al., J. Autoimmun., 11, 539-546 (1998); Weisbart, Int. J. Oncol., 25, 1867-1873 (2004)). In embodiments, the antibody or antigen-binding fragment described herein has the D3 IN substitution.
[0279] Antibody-payload conjugates described herein can be prepared with any 3E10 antibodies or antigen-fragments thereof, or any humanized 3E10 antibodies or antigen-fragments thereof, disclosed in the prior art. See, for example WO 2015/106290, 2016/033324, WO 2019/018426, and WO 2019/018428 (each of which is specifically incorporated by reference herein, in its entirety).
[0280] In embodiments, an antibody -payload conjugate provided herein comprises a humanized 3E10 antibody. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
[0281] In embodiments, the disclosure provides humanized antibodies, or antigen-binding fragments thereof, that incorporate any combination of the humanized VL and VH sequences disclosed here, as well as VL and VH sequences having sequence identity thereto, e.g., having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a VH or VL sequence described herein.
[0282] In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of any one of SEQ ID NOs: l, 13, or 71-84. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 1. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 13. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:71. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:72. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:73. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:74. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:75. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:76. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:77. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:78. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:79. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:80. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:81. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 82. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO:83. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a heavy chain having the sequence of SEQ ID NO: 84.
[0283] In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of any one of SEQ ID NOs:2, 14, 64-70, 103-112. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:2. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 14. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 64. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 65. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:66. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:67. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:68. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:69. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO:70. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 103. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 104. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 105. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 106. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 107. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 108. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 109. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 110. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 111. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VH having the sequence of SEQ ID NO: 112.
[0284] In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of any one of SEQ ID NOs:7, 20, or 91-102. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:7. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:20. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:91. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:92. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:93. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:94. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:95. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:96. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:97. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:98. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO:99. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO: 100. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO: 101. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a light chain having the sequence of SEQ ID NO: 102. [0285] In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of any one of SEQ ID NOs:8, 21, 85-90, or 113-121. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 8. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:21. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:85. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 86. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:87. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:88. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO:89. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NOVO. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 113. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 114. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 115. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 116. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 117. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 118. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 119. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 120. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, or three CDRs of a VL having the sequence of SEQ ID NO: 121.
[0286] In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of any one of SEQ ID NOs: 122-137. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 122. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 123. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 124. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 125. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 126. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 127. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 128. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 129. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 130. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 131. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 132. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 133. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 134. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 135. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO:136. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof comprising one, two, three, four, five, or six CDRs of an scFv having the sequence of SEQ ID NO: 137.
[0287] In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: l, 13, or 71-84. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 1. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 13. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:71. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:72. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:73. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:74. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:75. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:76. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:77. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%>, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:78. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%) identity to SEQ ID NO:79. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%>, at least about 80%>, at least about 85%>, at least about 90%>, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:80. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%) identity to SEQ ID NO:81. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%o, at least about 98%>, at least about 99%, or 100%) identity to SEQ ID NO:82. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:83. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a heavy chain sequence having at least about 75%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%o, at least about 96%, at least about 97%>, at least about 98%>, at least about 99%, or 100%> identity to SEQ ID NO:84.
[0288] In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs:7, 20, or 91-102. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:7. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:20. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:91. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:92. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:93. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:94. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:95. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:96. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:97. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:98. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:99. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 100. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 101. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a light chain sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 102.
[0289] In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 2, 14, 64-70, or 103-112. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:2. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 14. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:64. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:65. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:66. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:67. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:68. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:69. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:70. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 103. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 104. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 105. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 106. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 107. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 108. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 109. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 110. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:111. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VH sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 112.
[0290] In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to any one of SEQ ID NOs: 8, 21, 85-90, or 113-121. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:8. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:21. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:85. In embodiments, an antibody -payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 86. In embodiments, an antibody- payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:87. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:88. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:89. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NOVO. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 113. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 114. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 115. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 116. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 117. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 118. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO:119. In embodiments, an antibody -pay load conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 120. In embodiments, an antibody-payload conjugate provided herein comprises a 3E10 antibody or antigen-binding fragment thereof having a VL sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to SEQ ID NO: 121.
[0291] In embodiments, an antibody -payload conjugate provided herein comprises a humanized 3E10 antibody, or antigen-binding fragment thereof, comprising a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least about 97% identical to an amino acid sequence selected from the group consisting of 3E10-VL-hl (SEQ ID NO:85), 3E10- VL-h2 (SEQ ID NO:86), 3E10-VL-h3 (SEQ ID NO:87), 3E10-VL-h4 (SEQ ID NO:88), 3E10- VL-h5 (SEQ ID NO:89), and 3E10-VL-h6 (SEQ ID NOVO) and a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-hl (SEQ ID NO:64), 3E10-VH-h2 (SEQ ID NO:65), 3E10-VH-h3 (SEQ ID NO:66), 3E10-VH-h4 (SEQ ID NO:67), 3E10-VH-h5 (SEQ ID NO:68), 3E10-VH-h6 (SEQ ID NO:69), and 3E10-VH-h7 (SEQ ID NO:70).
[0292] In embodiments, the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-hl (SEQ ID NO:85). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-hl (SEQ ID NO:85). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-hl (SEQ ID NO:85). In embodiments, the sequence of the 3E10-VL is 3E10-VL-hl (SEQ ID NO:85).
[0293] In embodiments, the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h2 (SEQ ID NO:86). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h2 (SEQ ID NO:86). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h2 (SEQ ID NO:86). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h2 (SEQ ID NO:86).
[0294] In embodiments, the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h3 (SEQ ID NO:87). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h3 (SEQ ID NO:87). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h3 (SEQ ID NO:87). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h3 (SEQ ID NO:87).
[0295] In embodiments, the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h4 (SEQ ID NO:88). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h4 (SEQ ID NO:88). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h4 (SEQ ID NO:88). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h4 (SEQ ID NO:88).
[0296] In embodiments, the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h5 (SEQ ID NO:89). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h5 (SEQ ID NO:89). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h5 (SEQ ID NO:89). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h5 (SEQ ID NO:89).
[0297] In embodiments, the sequence of the 3E10-VL is at least about 97% identical to 3E10-VL-h6 (SEQ ID NO:90). In embodiments, the sequence of the 3E10-VL is at least about 98% identical to 3E10-VL-h6 (SEQ ID NO:90). In embodiments, the sequence of the 3E10-VL is at least about 99% identical to 3E10-VL-h6 (SEQ ID NO:90). In embodiments, the sequence of the 3E10-VL is 3E10-VL-h6 (SEQ ID NO:90).
[0298] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-hl (SEQ ID NO:64). In embodiments, the sequence of the 3E10-VH is 3E10-VH-hl (SEQ ID NO:64).
[0299] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h2 (SEQ ID NO:65). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h2 (SEQ ID NO:65).
[0300] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h3 (SEQ ID NO:66). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h3 (SEQ ID NO:66).
[0301] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h4 (SEQ ID NO:67). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h4 (SEQ ID NO:67).
[0302] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h5 (SEQ ID NO:68). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h5 (SEQ ID NO:68).
[0303] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h6 (SEQ ID NO:69). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h6 (SEQ ID NO:69).
[0304] In embodiments, the sequence of the 3E10-VH is at least about 95% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 96% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 97% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 98% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is at least about 99% identical to 3E10-VH-h7 (SEQ ID NO:70). In embodiments, the sequence of the 3E10-VH is 3E10-VH-h7 (SEQ ID NO:70).
[0305] In embodiments, an antibody -payload conjugate comprising a humanized 3E10 antibody, or antigen-binding fragment thereof, described herein includes a light chain (3E10-LC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-LC-hlm (SEQ ID NO:91), 3E10-LC-h2m (SEQ ID NO:92), 3E10-LC-h3m (SEQ ID NO:93), 3E10-LC-h4m (SEQ ID NO:94), 3E10-LC-h5m (SEQ ID NO:95), and 3E10-LC-h6m (SEQ ID NO:96) and a heavy chain (3E10-HC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-hlm (SEQ ID NO:71), 3E10-HC-h2m (SEQ ID NO: 72), 3E10- HC-h3m (SEQ ID NO:73), 3E10-HC-h4m (SEQ ID NO:74), 3E10-HC-h5m (SEQ ID NO:75), 3E10-HC-h6m (SEQ ID NO: 76), and 3E10-HC-h7m (SEQ ID NO: 77).
[0306] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-hlm (SEQ IDN0:91). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-hlm (SEQ ID NO:91). In embodiments, the sequence of the 3E10-LC is 3E10-LC-hlm (SEQ ID NO:91).
[0307] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h2m (SEQ IDNO:92). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h2m (SEQ ID NO:92). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h2m (SEQ ID NO:92).
[0308] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h3m (SEQ ID NO:93). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h3m (SEQ ID NO:93).
[0309] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h4m (SEQ ID NO:94). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h4m (SEQ ID NO:94).
[0310] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h5m (SEQ ID NO:95). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h5m (SEQ ID NO:95).
[0311] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h6m (SEQ IDNO:96). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h6m (SEQ ID NO:96). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h6m (SEQ ID NO:96).
[0312] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-hlm (SEQ ID NO:71). In embodiments, the sequence of the 3E10-HC is 3E10-HC-hlm (SEQ ID NO:71).
[0313] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h2m (SEQ ID NO:72). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h2m (SEQ ID NO:72). [0314] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h3m (SEQ ID NO:73). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h3m (SEQ ID NO:73).
[0315] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h4m (SEQ ID NO:74). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h4m (SEQ ID NO:74).
[0316] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h5m (SEQ ID NO:75). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h5m (SEQ ID NO:75).
[0317] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h6m (SEQ ID NO:76). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h6m (SEQ ID NO:76).
[0318] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h7m (SEQ ID NO:77). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h7m (SEQ ID NO:77).
[0319] In embodiments, an antibody-payload conjugate provided herein comprises a humanized 3E10 antibody, or antigen-binding fragment thereof, comprising a light chain (3E10- LC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-LC-hl (SEQ ID NO:97), 3E10-LC-h2 (SEQ ID NO:98), 3E10-LC-h3 (SEQ ID NO:99), 3E10-LC-h4 (SEQ ID NO: 100), 3E10-LC-h5 (SEQ ID NO: 101), and 3E10-LC-h6 (SEQ ID NO: 102) and a heavy chain (3E10-HC) comprising an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of 3E10-HC-hl (SEQ ID NO:78), 3E10-HC-h2 (SEQ ID NO:79), 3E10-HC- h3 (SEQ ID NO:80), 3E10-HC-h4 (SEQ ID NO:81), 3E10-HC-h5 (SEQ ID NO:82), 3E10-HC-h6 (SEQ ID NO:83), and 3E10-HC-117 (SEQ ID NO:84).
[0320] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-hl (SEQ ID NO:97. In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-hl (SEQ ID NO:97). In embodiments, the sequence of the 3E10-LC is 3E10-LC-hl (SEQ ID NO:97).
[0321] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h2 (SEQ ID NO:98). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h2 (SEQ ID NO:98). [0322] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h3 (SEQ ID NO:99). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h3 (SEQ ID NO:99).
[0323] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h4 (SEQ ID NO: 100). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h4 (SEQ ID NO: 100).
[0324] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h5 (SEQ ID NO: 101). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h5 (SEQ ID NO: 101).
[0325] In embodiments, the sequence of the 3E10-LC is at least about 95% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is at least about 96% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is at least about 97% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is at least about 98% identical to 3E10-LC-h6 (SEQ ID NO:102). In embodiments, the sequence of the 3E10-LC is at least about 99% identical to 3E10-LC-h6 (SEQ ID NO: 102). In embodiments, the sequence of the 3E10-LC is 3E10-LC-h6 (SEQ ID NO: 102).
[0326] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-hl (SEQ ID NO:78). In embodiments, the sequence of the 3E10-HC is 3E10-HC-hl (SEQ ID NO:78).
[0327] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h2 (SEQ ID NO:79). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h2 (SEQ ID NO:79).
[0328] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-113 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h3 (SEQ ID NO:80). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h3 (SEQ ID NO:80).
[0329] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h4 (SEQ ID NO:81). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h4 (SEQ ID NO:81).
[0330] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h5 (SEQ ID NO:82). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h5 (SEQ ID NO:82).
[0331] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h6 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h6 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-116 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h6 (SEQ ID NO:83). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h6 (SEQ ID NO:83). In some aspects, the sequence of the 3E10-HC is 3E10-HC-h6 (SEQ ID NO:83).
[0332] In embodiments, the sequence of the 3E10-HC is at least about 95% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is at least about 96% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is at least about 97% identical to 3E10-HC-h7 (SEQ ID NO: 84). In embodiments, the sequence of the 3E10-HC is at least about 98% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is at least about 99% identical to 3E10-HC-h7 (SEQ ID NO:84). In embodiments, the sequence of the 3E10-HC is 3E10-HC-h7 (SEQ ID NO:84).
[0333] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein has CDR sequences corresponding to those in the parent 3E10 antibody, optionally including a D31N amino acid substitution in the VH CDR1. Accordingly, in embodiments, a humanized 3E10 antibody or antigen-binding fragment thereof includes a light chain variable domain (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3 (SEQ ID NO: 11), a heavy chain variable domain (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDRla (SEQ ID NO: 16), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).
[0334] In embodiments, an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes CDR sequences from a variant humanized 3E10 antibody that includes a D31N amino acid substitution in the VH CDR1 (SEQ ID NO: 15).
[0335] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than seven amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
[0336] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than ten amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10- VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NON 1), 3E10-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
[0337] In embodiments, an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than nine amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
[0338] In embodiments, an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than eight amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
[0339] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than seven amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO: 9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO:11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
[0340] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than six amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10- VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NO:11), 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
[0341] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than five amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
[0342] In embodiments, an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than four amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
[0343] In embodiments, an antibody -payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than three amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NON).
[0344] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than two amino acid substitutions, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO:11), 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
[0345] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than one amino acid substitution, relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10- VL-CDR2 (SEQ ID NONO), 3E10-VL-CDR3 (SEQ ID NO:11), 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
[0346] Accordingly, in embodiments, an antibody-payload conjugate described herein can comprise a humanized 3E10 antibody or antigen-binding fragment thereof includes a light chain variable domain (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of 3E10-VL-CDR1 (SEQ ID NO: 9), a VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2 (SEQ ID NO: 10), a VL CDR3 comprising the amino acid sequence of 3E10- VL-CDR3 (SEQ ID NO: 11), a heavy chain variable domain (VH) CDR1 comprising the amino acid sequence of 3E10-VH-CDR1 D31N (SEQ ID NO: 15), a VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2 (SEQ ID NO: 4), and a VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3 (SEQ ID NO: 5).
[0347] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a set of complementarity determining regions (CDRs) collectively having no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, relative to the CDR sequences of 3E10-D31N variant (SEQ ID NOs: 15- 18 and 22-24), selected from, but not limited to, a G to S substitution at position 5 of VH CDR2, a T to S substitution at position 14 of VH CDR2, an S to T substitution at position 5 of VL CDR1, an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2.
[0348] Accordingly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.1 (SEQ ID NO: 26) or 3E10-VH-CDR2.2 (SEQ ID NO: 27). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l l), and VH CDRs 1 and 3 (SEQ ID NOs:3 and 5) according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ ID NOs: 15 and 18) according to the 3E10-D3 IN variant.
[0349] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.1 (SEQ ID NO: 28) or 3E10-VL-CDR1.2 (SEQ ID NO: 29). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs: 10 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CRDs 1 -3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10- D31N variant.
[0350] Similarly, in embodiments, an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.1 (SEQ ID NO: 30). In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 11), and VH CDRs 1-3 (SEQ ID NOs: 3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:22 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18)according to the 3E10-D31N variant.
[0351] While some of the amino acid substitutions described above are fairly conservative substitutions — e.g., an S to T substitution at position 5 of VL CDR1 — other substitutions are to amino acids that have vastly different properties — e.g., an M to L substitution at position 14 of VL CDR1, an H to A substitution at position 15 of VL CDR1, and an E to Q substitution at position 6 of VL CDR2. This suggests, without being bound by theory, that at least these positions within the 3E10 CDR framework are tolerant to other amino acid substitutions.
[0352] Accordingly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.3 (SEQ ID NO: 31). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 1 and 3 (SEQ ID NOs:3 and 5) according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragmentthereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ ID NOs: 15 and 18) according to the 3E10- D31N.
[0353] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDR1.3 (SEQ ID NO: 32). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs: 10 and 11), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10- D3 IN variant.
[0354] Similarly, in embodiments, an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof, includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.2 (SEQ ID NO: 33). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:22 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10- D31N variant.
[0355] Accordingly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDR1.C1 (SEQ ID NO: 34), 3E10-VH-CDRl.c2 (SEQ ID NO: 35), 3E10-VH-CDRl.c3 (SEQ ID NO: 36), 3E10-VH-CDRl.c4 (SEQ ID NO: 37), or 3E10-VH- CDRl.c5 (SEQ ID NO: 38). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 2 and 3 (SEQ ID NOs:4 and 5) according to the parent 3E10 antibody.
[0356] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2.cl (SEQ ID NO: 39), 3E10-VH-CDR2.c2 (SEQ ID NO: 40), or 3E10-VH-CDR2.c3 (SEQ ID NO: 41). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 -3 (SEQ ID NOs:9-l l), and VH CDRs 1 and 3 (SEQ ID NOs:3 and 5) according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ IDNOs: 15 and 18) according to the 3E10-D3 IN variant.
[0357] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3.cl (SEQ ID NO: 42), 3E10-VH-CDR3.c2 (SEQ ID NO: 43), or 3E10-VH-CDR3.c3 (SEQ ID NO: 44). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l l), and VH CDRs 1 and 2 (SEQ ID NOs:3 and 4) according to the parent 3E10 antibody. In embodiments, the 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 2 (SEQ ID NOs: 15 and 17) according to the 3E10-D3 IN variant.
[0358] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRl.cl (SEQ ID NO: 45), 3E10-VL-CDRl.c2 (SEQ ID NO: 46), 3E10-VL-CDR1.C3 (SEQ ID NO: 47), 3E10-VL-CDRl.c4 (SEQ ID NO: 48), 3E10-VL- CDRl.c5 (SEQ ID NO: 49), or 3E10-VL-CDRl.c6 (SEQ ID NO: 50). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:10 and 1 1), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CRDs 1-3 (SEQ ID NOs: 15, 17, 18) according to the 3E10-D31N variant.
[0359] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2.cl (SEQ ID NO: 51). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:22 and 24), and VH CDRs 1-3 (SEQ ID NOs:15, 17 and 18) according to the 3E10-D3 IN variant. [0360] Similarly, in some aspects, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3.cl (SEQ ID NO: 52), 3E10-VL-CDR3.c2 (SEQ ID NO: 53), 3E10-VL-CDR3.c3 (SEQ ID NO: 54), 3E10-VL-CDR3.c4 (SEQ ID NO: 55), 3E10-VL- CDR3.c5 (SEQ ID NO: 56), or 3E10-VL-CDR3.c6 (SEQ ID NO: 57). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:9 and 10), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:22 and 23), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10-D31N variant.
[0361] It is also contemplated that an antibody-payload conjugate comprising a humanized
3E10 antibody or antigen-binding fragment thereof, as described herein, includes no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 CDR amino acid substitutions of the CDR amino acid substitutions described above. Further examples of 3E10 variant CDR sequences are described herein (SEQ ID NOs:58-63).
[0362] Accordingly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR1 comprising the amino acid sequence of 3E10-VH-CDRlm (SEQ ID NO: 58). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 2 and 3 (SEQ ID NOs:4 and 5) according to the parent 3E10 antibody.
[0363] Similarly, in embodiments, an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR2 comprising the amino acid sequence of 3E10-VH-CDR2m (SEQ ID NO: 59). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 1 and 3 (SEQ ID NOs: 3 and 5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 3 (SEQ ID NOs: 15 and 18) according to the 3E10-D3 IN variant.
[0364] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VH CDR3 comprising the amino acid sequence of 3E10-VH-CDR3m (SEQ ID NO: 60). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:9-l 1), and VH CDRs 1 and 2 (SEQ ID NOs:3 and 4) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1-3 (SEQ ID NOs:22-24), and VH CDRs 1 and 2 (SEQ ID NOs: 15 and 17) according to the 3E10-D3 IN variant.
[0365] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR1 comprising the amino acid sequence of 3E10-VL-CDRlm (SEQ ID NO: 61). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs: 10 and 11), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 2 and 3 (SEQ ID NOs:23 and 24), and VH CDRs 1-3 (SEQ ID NOs:15,17, and 18) according to the 3E10-D31N variant.
[0366] Similarly, in embodiments, an antibody -pay load conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR2 comprising the amino acid sequence of 3E10-VL-CDR2m (SEQ ID NO: 62). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs:9 and 1 1), and VH CDRs 1 -3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 3 (SEQ ID NOs: 22 and 24), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10-D3 IN variant.
[0367] Similarly, in embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof includes VL CDR3 comprising the amino acid sequence of 3E10-VL-CDR3m (SEQ ID NO: 63). In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:9 and 10), and VH CDRs 1-3 (SEQ ID NOs:3-5) according to the parent 3E10 antibody. In embodiments, the humanized 3E10 antibody or antigen-binding fragment thereof further includes VL CDRs 1 and 2 (SEQ ID NOs:9 and 10), and VH CDRs 1-3 (SEQ ID NOs: 15, 17 and 18) according to the 3E10-D3 IN variant. [0368] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a light chain variable domain (3E10-VL) comprising an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of 3E10-VL-hl (SEQ ID NO:85), 3E10- VL-h2 (SEQ ID NO:86), 3E10-VL-h3 (SEQ ID NO:87), 3E10-VL-h4 (SEQ ID NO:88), 3E10- VL-h5 (SEQ ID NO:89), and 3E10-VL-h6 (SEQ ID NO: 90), where the light chain variable domain (3E10-VL) further comprises one or more amino acid residues selected from proline (Pro) at position 15, threonine (Thr) at position 22, tyrosine (Tyr) at position 49, Thr at position 74, asparagine (Asn) at position 76, alanine (Ala) at position 80, Asn at position 81, Thr at position 83, Asn at position 85, and valine (Vai) at position 104, of the 3E10-VL according to Kabat numbering, and a set of 3E10-VL CDRs collectively having no more than 6 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NOTO), 3E10-VL-CDR3 (SEQ ID NO: 11), and where the antibody includes a set of 3E10-VL CDRs collectively having no more than 6 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID N0:9), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
[0369] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 5 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NOTO), 3E10-VL-CDR3 (SEQ ID NO: 11).
[0370] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 4 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NO:9), 3E10-VL-CDR2 (SEQ ID NOTO), 3E10-VL-CDR3 (SEQ ID NOT 1).
[0371] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 3 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
[0372] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 2 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
[0373] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising no more than 1 amino acid substitution relative to the set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
[0374] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VL CDRs comprising a set of CDRs having the amino acid sequences of 3E10-VL-CDR1 (SEQ ID NON), 3E10-VL-CDR2 (SEQ ID NO: 10), 3E10-VL-CDR3 (SEQ ID NO: 11).
[0375] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a lysine (Lys) residue at position 49 of the 3E10-VL according to Kabat numbering.
[0376] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a glutamic acid (Glu) residue at position 81 of the 3E10-VL according to Kabat numbering.
[0377] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a proline (Pro) residue at position 15 of the 3E10-VL according to Kabat numbering.
[0378] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a valine (Vai) residue at position 104, of the 3E10-VL according to Kabat numbering. [0379] In embodiments, an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof described herein includes a heavy chain variable domain (3E10-VH) comprising an amino acid sequence that is at least about 90% identical to an amino acid sequence selected from the group consisting of 3E10-VH-hl (SEQ ID NO:64), 3E10- VH-h2 (SEQ ID NO:65), 3E10-VH-h3 (SEQ ID NO:66), 3E10-VH-h4 (SEQ ID NO:67), 3E10- VH-h5 (SEQ ID NO:68), 3E10-VH-h6 (SEQ ID NO:69), and 3E10-VH-h7 (SEQ ID NO:70), where the heavy chain variable domain (3E10-VH) further comprises one or more amino acid residues selected from glutamine (Gin) at position 13, leucine (Leu) at position 18, arginine (Arg) at position 19, glycine (Gly) at position 42, serine (Ser) at position 49, Ser at position 77, tyrosine (Tyr) at position 79, Asn at position 82, Ala at position 84, Vai at position 89, leucine (Leu) at position 108, Vai at position 109, and Ser at position 113, of the 3E10-VH according to Kabat numbering, and where the antibody includes a set of 3E10-VH CDRs collectively having no more than 6 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO:5).
[0380] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 5 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO 5).
[0381] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 4 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO:5).
[0382] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 3 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NO:4), and 3E10-VH- CDR3 (SEQ ID NO:5). [0383] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes set of 3E10-VH CDRs comprising no more than 2 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E1O-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH- CDR3 (SEQ ID NO:5).
[0384] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 1 amino acid substitution relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO:15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH- CDR3 (SEQ ID NO 5)
[0385] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof includes a set of 3E10-VH CDRs comprising no more than 5, 4, 3, 2, or 1 amino acid substitutions relative to the set of CDRs having the amino acid sequences of 3E10-VH-CDR1 D31N (SEQ ID NO: 15), 3E10-VH-CDR2 (SEQ ID NON), and 3E10-VH-CDR3 (SEQ ID NO:5).
[0386] In one aspect, the present disclosure provides an antibody -payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with an arginine (Arg) residue at position 18 of the 3E10-VH according to Kabat numbering.
[0387] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a (Lys) residue at position 19 of the 3E10-VH according to Kabat numbering.
[0388] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with an alanine (Ala) residue at position 49 of the 3E10-VH according to Kabat numbering.
[0389] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a glutamine (Gin) residue at position 13, of the 3E10-VH according to Kabat numbering. [0390] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a leucine (Leu) residue at position 108, of the 3E10-VH according to the Kabat numbering.
[0391] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a valine (Vai) residue at position 109, of the 3E10-VH according to Kabat numbering.
[0392] In one aspect, the present disclosure provides an antibody -payload conjugate comprising a humanized antibody or antigen-binding fragment thereof with a serine (Ser) residue at position 113, of the 3E10-VH according to Kabat numbering.
[0393] In embodiments, the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof with a fragment crystallizable (Fc) region.
[0394] In embodiments, the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof with an Fc region selected from a human IgGl Fc, a human IgG2a Fc, a human IgG2b Fc, a human IgG3 Fc, and a human IgG4 Fc.
[0395] In embodiments, the present disclosure provides an antibody-payload conjugate comprising humanized 3E10 antibodies or variants thereof, or antigen-binding fragments thereof comprising a heavy chain constant domain (CH).
[0396] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof comprises an Fc region selected from a human yl CHI, a human γ2 CHI, a human γ3 CHI, and a human γ4 CHI.
[0397] In embodiments, the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or antigen-binding fragment thereof comprising a light chain constant domain (CL).
[0398] In one aspect, the present disclosure provides an antibody-payload conjugate comprising a humanized 3E10 antibody or variant comprising an Fc region selected from the group consisting of a human L CL and a human K CL. [0399] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof comprising a combination of a light chain variable domain (VL) and a heavy chain variable domain (VH) selected from 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-hl (SEQ ID NO:64), 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-h2 (SEQ ID NO:65), 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-h3 (SEQ ID NO:66), 3E10-VL-hl (SEQ ID NO:85) and 3E10-VH-h4 (SEQ ID NO:67), 3E10-VL-h2 (SEQ ID NO:86) and 3E10-VH-hl (SEQ ID NO:64), 3E10-VL-h2 (SEQ ID NO:86) and 3E10-VH-h2 (SEQ ID NO:65), 3E10-VL- h3 (SEQ ID NO:87) and 3E10-VH-hl (SEQ ID NO:64), 3E10-VL-h5 (SEQ ID NO:89) and 3E10- VH-h5 (SEQ ID NO:68), 3E10-VL-h5 (SEQ ID NO:89) and 3E10-VH-h6 (SEQ ID NO:69), 3E10- VL-h6 (SEQ ID NO:90) and 3E10-VH-h5 (SEQ ID NO:68), and 3E10-VL-h6 (SEQ ID NO:90) and 3E10-VH-h6 (SEQ ID NO:69).
[0400] In embodiments, an antibody-payload conjugate comprising the humanized 3E10 antibody or antigen-binding fragment thereof comprises a combination of a light chain variable domain (VL) of 3E10-VL-h6 (SEQ ID NO:90) and a heavy chain variable domain (VH) of 3E10- VH-h6 (SEQ ID NO:69).
[0401] Antibodies useful in the compositions, conjugates, and methods described herein include whole immunoglobulin (i.e., an intact antibody) of any class, fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domains differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of the variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. Therefore, the antibodies typically contain at least the CDRs necessary to maintain DNA binding. [0402] The 3E10 antibody is typically a monoclonal 3E10, or a variant, derivative, fragment, fusion, or humanized form thereof that binds the same or different epitope(s) as 3E10.
[0403] A deposit according to the terms of the Budapest Treaty of a hybridoma cell line producing monoclonal antibody 3E10 was received on September 6, 2000, and accepted by, American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110-2209, USA, and given Patent Deposit Number PTA-2439. Thus, the antibody can have the same or different epitope specificity as monoclonal antibody 3E10 produced by ATCC No. PTA 2439 hybridoma. The antibody can have the paratope of monoclonal antibody 3E10. The antibody can be a single chain variable fragment of 3E10, or a variant, e.g., a conservative variant thereof. For example, the antibody can be a single chain variable fragment of 3E10 (3E10 Fv), or a variant thereof.
[0404] Additionally, or alternatively, the heavy chain complementarity determining regions (CDRs) can be defined according to the IMGT system. The complementarity determining regions (CDRs) as identified by the IMGT system include CDR Hl.3 (original sequence): GFTFSDYG (SEQ ID NO:989); CDR Hl.4 (with D31N mutation): GFTFSNYG (SEQ ID NO:990); CDR H2.2: ISSGSSTI (SEQ ID NO:991) and variant ISSSSSTI (SEQ ID NO:992); CDR H3.2: ARRGLLLDY (SEQ ID NO:993).
[0405] Additionally, or alternatively, the light chain complementarity determining regions (CDRs) can be defined according to the IMGT system. The complementarity determining regions (CDRs) as identified by the IMGT system include CDR LI.2 KSVSTSSYSY (SEQ ID NO:994) and variant KTVSTSSYSY (SEQ IDNO:995); CDRL2.2: YAS; CDRL3.2: QHSREFPWT (SEQ ID NO: 996).
[0406] The disclosed compositions, conjugates, and methods typically utilize antibodies that maintain the ability to penetrate cells, and optionally nuclei. The mechanisms of cellular internalization by autoantibodies are diverse. Some are taken into cells through electrostatic interactions or FcR-mediated endocytosis, while others utilize mechanisms based on association with cell surface myosin or calreticulin, followed by endocytosis (Ying-Chyi et al., Eur J Immunol 38, 3178-3190 (2008), Yanase et al., J Clin Invest 100, 25-31 (1997)). The 3E10 antibodies and antigen-binding fragments thereof can transit cellular membranes via an equilibrative nucleoside (ENT) transporter. In embodiments, 3E10 transits cellular membranes via an ENT1, ENT2, ENT3 or ENT4 transporter (See, e.g., WO 2015/106290 Al and WO 2016/033324 Al, each of which is incorporated by reference herein, in its entirety). In embodiments, 3E10 penetrates cells in an Fc- independent mechanism (as evidenced by the ability of 3E10 fragments lacking an Fc to penetrate cells) but involves presence of the nucleoside transporter ENT2 (Weisbart et al., Sci Rep 5: 12022. doi: 10.1038/srepl2022. (2015), Zack et al., J Immunol 157, 2082-2088 (1996), Hansen et al., J Biol Chem 282, 20790-20793 (2007)). Thus, in embodiments, the antibodies utilized in the disclosed compositions, conjugates, and methods are ones that penetrates cells in an Fc- independent mechanism and involve the presence of the nucleoside transporter ENT2.
[0407] Mutations in 3E10 that interfere with its ability to bind DNA can render the antibody incapable of nuclear penetration. Thus, typically the disclosed variants and humanized forms of the antibody maintain the ability to bind nucleic acids, particularly DNA. In addition, 3E10 scFv has previously been shown capable of penetrating into living cells and nuclei in an ENT2-dependent manner, with efficiency of uptake impaired in ENT2-deficient cells (Hansen, et al., J. Biol. Chem. 282, 20790-20793 (2007)). Thus, in embodiments, the disclosed variants and humanized forms of the antibody maintain the ability penetrate into cell nuclei in an ENT2- dependent manner.
[0408] As discussed in US 2021/0054102 and US 2021/0137960, some humanized 3E10 variant were found to penetrate cell nuclei more efficiently than the original murine 3E10 (D31N) di-scFv, while others were found to have lost the ability to penetrate nuclei. In particular, variants 10 and 13 penetrated nuclei very well compared to the murine antibody.
[0409] Potential bipartite nuclear localization signals (NLS) in humanized 3E10 VL have been identified and may include part or all of the following sequences:
RASKSVSTSSYSYMHWYQQKPGQPPKLLIKY (SEQ ID NO: 138);
RASKTVSTSSYSYMHWYQQKPGQPPKLLIKY (SEQ ID NO: 139); or RVTITCRASKSVSTSSYSYMHWYQQKPGKAPKL (SEQ ID NO: 140).
[0410] An example consensus NLS can be, or include, (X)RASKTVSTSSYSYMHWYQQKPGQPPKLL(X)KY (where (X) = any residue, but preferentially is a basic residue (R or K) (SEQ ID NO: 141) or a variant thereof with at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 percent sequence identity to SEQ ID NO: 142. [0411] Thus, in some embodiments, particularly where nuclear importation is important, the disclosed antibodies may include the sequence of any one of SEQ ID NOs: 138-142, or fragments and variants thereof (e.g., at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% amino acid sequence identity with any one of SEQ ID NOs: 138-142) that can translocate into the nucleus of a cell.
[0412] Presence of an NLS indicates that a humanized 3E10 antibody or antigen binding fragment thereof may cross the nuclear envelope via the nuclear import pathway. In some embodiments, the NLS improves importation by interacting with one or more members of the import pathway. Thus, in some embodiments, the NLS can bind to importin- , an importin- β/importin-α heterodimer, or a combination thereof.
[0413] In some embodiments, the disclosed compositions and methods utilize humanized 3E10 antibodies and ENT2-binding fragments thereof that maintain the ability to bind nucleic acids such as DNA, RNA.
[0414] The Examples below illustrate molecular modeling of wild type 3E10 sequences and additional 3E10 variants. Molecular modeling of 3E10 (Pymol) revealed a putative Nucleic Acid Binding pocket (NAB 1) (See, e g., Figures 11 A and 1 IB, and illustrated with underlining the sequences below).
WT HEAVY CHAIN scFv SEQUENCE
E VQLVESGGGL VKPGGSRKLS CAASGFTFSD YGMHWVRQAP EKGLEWVAYI SSGSSTIYYA DTVKGRFTIS RDNAKNTLFL QMTSLRSEDT AMYYCARRGL LLDYWGQGTT LTVS (SEQ ID NO: 148)
LIGHT CHAIN scFv SEQUENCE
D IVLTQSPASL AVSLGQRATI SCRASKSVST SSYSYMHWYQ QKPGQPPKLL IKYASYLESG VPARFSGSGS GTDFTLNIHP VEEEDAATYY CQHSREFPWT FGGGTKLEIK RADAAPGGGG SGGGGSGGGGS (SEQ ID NO: 149)
[0415] In some embodiments, the disclosed humanized 3E10 antibodies include some or all of the underlined NAB1 sequences. In some embodiments, the humanized 3E10 antibodies include a variant sequence that has an altered ability of bind nucleic acids. In some embodiments, the mutations (e.g., substitutions, insertions, and/or deletions) in the NAB1 improve binding of the antibody to nucleic acids such as DNA, RNA, or a combination thereof. In some embodiments, the mutations are conservative substitutions. In some embodiments, the mutations increase the cationic charge of the NAB1 pocket.
[0416] As discussed and exemplified herein, mutation of aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).
[0417] Additional example variants include mutation of aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R), which modeling indicates expands cationic charge, or lysine (3E10-D3 IK) which modeling indicates changes charge orientation. Thus, in some embodiments, the 3E10 binding protein includes a D31R or D3 IK substitution.
[0418] Additional example variants include mutation of arginine (R) 96 to asparagine (N), and/or serine (S) 30 to aspartic acid (D) alone or in combination with D3 IN, D31R, or D3 IK.
[0419] Molecular modeling of 3E10 (Pymol) revealed a putative Nucleic Acid Binding pocket (NAB1) (Figures 11A-1 IB). Mutation of aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D31N).
[0420] Mutation of aspartic acid at residue 31 of CDR1 to arginine (3E10-D31R), further expanded the cationic charge while mutation to lysine (3E10-D31K) changed charge orientation (Figure 11A).
[0421] NAB 1 amino acids predicted from molecular modeling have been underlined in the heavy and light chain sequences above. Figure 1 IB is an illustration showing molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues illustrated with punctate dots.
[0422] All of the sequences disclosed herein having the residue corresponding with R96 are expressly disclosed with R96N substitution.
[0423] All of the sequence disclosed herein having the residue corresponding to S30 are expressly disclosed with S30D.
[0424] Any of the substitutions can be included in any combination. The sequence having two or three substitutions at any combination of residues 31, 30, and 96 are expressly provided. [0425] In particular embodiments, the sequence has 3 IN, 3 IK, or 31R alone or in combination with 30D, and without the R96N substitution. Thus, in some embodiments, the residue corresponding to 96 is not N, and in more specific embodiments remains R.
B. Linkers
[0426] In some aspects, the antibody-oligonucleotide conjugates (AOCs) provided herein comprises a linker. The linker (L) described herein can be used to link or conjugate the 3E10 antibody or antigen-binding fragment thereof to an oligonucleotide, e.g., an siRNA or antisense oligonucleotide. The term “linker” as used herein includes, without limitation, any known linker for use in antibody-oligonucleotide-conjugates known in the art. In some aspects, the AOC comprises, one, two, three, four, or more linkers.
1. Conjugation Sites and Methods
[0427] In some embodiments, one or more amino acids suitable conjugation of a 3E10 antibody or antigen-binding fragment thereof provided herein are selected from lysine, cysteine, histidine, arginine, aspartic acid, glutamine, serine, threonine and tyrosine. In some embodiments, one or more amino acids suitable for conjugation are introduced by substitution of one or more amino acids in the 3E10 antibody or antigen-binding fragment thereof. In some embodiments, the one or more conjugated amino acids are lysine or arginine, and conjugation is conducted via amine conjugation. In some embodiments, one or more conjugated amino acids are glutamine (Gin) and conjugation is conducted via transglutaminase (TGase) mediated enzymatic conjugation. In some embodiments, one or more conjugated amino acids are cysteine (Cys) and conjugation is conducted via thiol conjugation. For example, NHS-PEG reagent can be used to modify primary amines in a 3E10 antibody or antigen-binding fragment thereof provided herein.
[0428] In some embodiments, a composition comprising an AOC as described herein has an average DAR of at least 4 (an average of at least four drug moieties attached to each antibody). In some embodiments, such a composition has an average DAR of at least 6. In some embodiments, such a composition has an average DAR of at least 8. In some embodiments, such a composition has an average DAR of at least 10. In some embodiments, such a composition has an average DAR of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more. [0429] In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of no more than 4.
[0430] In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 4. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 2 to 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 4. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 3 to 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 6. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 4 to 12. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 6 to 8. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 6 to 10. In some embodiments, a composition comprising an AOC as described herein has an average DAR of from 6 to 12.
[0431] In particular, a method of site-specific conjugation is by means of transglutaminase.
Transglutaminases (TGases) which also include bacterial transglutaminase (BTG) are a family of enzymes which catalyse the formation of a covalent bond between the y-carbonyl-amide group of glutamines and the primary amine group of lysines.
[0432] A peptide or antibody can be a substrate for transglutaminase according to the methods of the present disclosure. Thus, in some embodiments, the peptide or antibody contains a Gin or a Lys residue, and in particular a Gin residue. In some embodiments, the peptide or antibody is not a transglutaminase substrate, so one or more Gin or Lys residues, and in particular Gin residues, are inserted into the peptide or antibody sequence to make the peptide a substrate for transglutaminase. In embodiments, a Gin or Lys residue may be inserted at any position in the peptide or antibody sequence, however, it is preferably inserted at a position where the physiological properties, such as the therapeutic activity of the peptide is not affected to a degree where the peptide is not useful anymore, e.g., in a therapeutic intervention. Insertions of amino acid residues in peptides can be brought about by standard techniques known to persons skilled in the art, such as post-translational chemical modification or transgenic techniques, as described, for example, in US Patent No. 11,123,439 and US 2016/0355859, the contents of which are hereby incorporated by reference.
[0433] Since such transglutaminases also accept substrates other than lysine as amine donor, they are used to modify proteins including antibodies at suitable acceptor glutamines (Josten et al., J. Immunol. Methods 240, 47-54 (2000); Mindt et al., Bioconjugate Chem. 19, 271- 278 (2008); Dennler et al., in Antibody Drug Conjugates (Ducry, L , Ed.), pp 205-215, Humana Press. (2013), the contents of which are incorporated herein by reference). Transglutaminases have been used for the conjugation of drugs to antibodies containing artificial glutamine tags which are acceptor glutamine residues which have been introduced into the antibody by genetic engineering (Strop et al., Chem. Biol. 20, 161-167 (2013)). Furthermore, the conserved glutamine residue Q295 (Kabat EU numbering) of the constant region of the heavy chain of antibodies is the only y-carbonyl-amide donor for the bacterial transglutaminase (EC 2.3.2.13) in the backbone of aglycosylated IgGl molecules, and is thus an acceptor glutamine, whereas no acceptor glutamine is present in the backbone of IgGl when the antibody has been glycosylated at position N297 (Kabat EU numbering) of the heavy chain. In summary, bacterial transglutaminase can be used for the conjugation of an amine-donor substrate, for example a drug-linker construct, at an acceptor glutamine residue of an antibody. Such acceptor glutamines can be introduced by engineering of the antibody by mutations or by the generation of aglycosylated antibodies. Such aglycosylated antibodies can be introduced by deglycosylation using N-glycosidase F (PNGase F) or by mutation of N297 of the glycosylation site of the heavy chain (Kabat EU numbering) to any other amino acid except N. The enzymatic conjugation of such aglycosylated antibodies using bacterial transglutaminase has been described for aglycosylated antibody variants containing the mutations N297D, N297Q or N297S (see U.S. Pat Nos. US 9,764,038 and US 9,764,038, the contents of which are incorporated by reference). The enzymatic conjugation of such aglycosylated antibodies by means of transglutaminase generally affords AOCs having a drug (oligonucleotide) antibody ratio (DAR or OAR) of 2, in which both heavy chains are specifically functionalized at position Q295 (Kabat EU numbering). Only mutation N297Q of the heavy chain affords an additional conjugation site per heavy chain. The conjugation of such variants leads to AOCs having a DAR of 4, in which both heavy chains are specifically functionalized at positions Q295 and Q297.
[0434] In embodiments, the chemical modification strategy utilized to create antibody- payload conjugates described herein is lysine conjugation. Lysine residues in proteins, e.g., antibodies, possess a primary amine group (-NH2) in their side chains, making them suitable targets for chemical modification. This primary amine group can react with various chemical reagents, including small molecules or polymers (e.g., cleavable and non-cleavable linkers).
[0435] In embodiments, lysine conjugation can be either site-specific or random. In site- specific conjugation, specific lysine residues within a protein, e.g., antibody, can be targeted ensuring precise control over the modification. In contrast, random conjugation involves modifying lysine residues without selectivity.
[0436] However, tryptic peptide mapping of 3E10 conjugated at lysine residues has shown that V region lysines in both VL and VH were observed to be modified and, in fact, VL K53 is the most modified lysine. Furthermore, the humanization trials described in WO 2023/168352, incorporated herein by reference in its entirety, demonstrated that any modification to VL K53 negatively impacted the immunoreactivity to nucleic acids of the humanized 3E10-D31N monoclonal antibody (V66).
[0437] In embodiments, the conjugation strategy is selected from one of the following:
a. Lysine Attachment
[0438] In some embodiments of AOCs described herein, the linker moiety is conjugated to the antibody or antigen binding fragment therein through an amine linkage at one or more surface-exposed lysine residues on the antibody or antigen binding fragment thereof. Generally, lysine conjugation is a random process, with respect to which lysines are conjugated to the liker- payload. However, some preference for conjugation at certain lysines can occur due to the context of the primary, secondary, ternary, and/or quaternary structure surrounding a particular lysine residue. Many different chemistries are known in the art for attaching payloads to proteins at lysine groups. For example, activated esters on the drug-linker complexes, often O-succinimide reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters, can react with the antibody lysine residues and achieve conjugation via amide bonds, or stable amidine bonds can be generated on an antibody by the reaction of imido ester compounds, such as Traut’s reagent, with antibody lysine residues. Further description of lysine conjugation techniques are described, for example, in Walker, J.M., et al., “Antibody-drug conjugates,” Humana Press (2013); Bhat, A.S., et al., “The next step in homogenous bioconjugate development: optimizing payload placement and conjugate composition,” BioProcess International (2014); and Jain, N., et al., “Current ADC linker chemistry,” Pharm Res., 32:3526-40 (2015), the disclosure of which are incorporated herein by reference, in their entireties, for all purposes. [0439] Accordingly, in some embodiments, the present disclosure provides pharmaceutical compositions comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L- Pr)q, where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein, L is a linker as described herein, P is an oligonucleotide moiety as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, in which L is conjugated to A through lysine moieties.
[0440] Advantageously, because the structural regions of antibodies contain many lysine residues, lysine conjugation can be used to generate AOC molecules with a high drug to antibody ratio (DAR). Accordingly, in some embodiments, a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein will have an average DAR of at least 4 (an average of at least four drug moieties attached to each antibody). In some embodiments, such a composition will have an average DAR of at least 6. In some embodiments, such a composition will have an average DAR of at least 8. In some embodiments, such a composition will have an average DAR of at least 10. In some embodiments, such a composition will have an average DAR of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more.
[0441] Accordingly, in some embodiments, a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 1 and q is at least 4. In some embodiments, r is 1 and q is at least 6. In some embodiments, r is 1 and q is at least 8. In some embodiments, r is 1 and q is at least 10. In some embodiments, r is 1 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more. In some embodiments, a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 2 (e.g., the linker is a branched linker) and q is at least 2. In some embodiments, r is 2 and q is at least 3. In some embodiments, r is 2 and q is at least 4. In some embodiments, r is 2 and q is at least 5. In some embodiments, r is 2 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more. In yet other embodiments, a composition comprising an AOC where lysine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, the linker is highly branched, e.g., r is at least 3. In some embodiments, r is at least 4. In some embodiments, r is at least 5, 6, 7, 8, or more. [0442] In some aspects, lysine conjugation can be either site-specific or random. In site- specific conjugation, specific lysine residues within a protein, e.g., antibody, can be targeted ensuring precise control over the modification. In contrast, random conjugation involves modifying lysine residues without selectivity.
[0443] However, as described in Example 5 and Figures 22A-22C, tryptic peptide mapping of 3E10 conjugated at lysine residues showed that V region lysines in both VL and VH were observed to be modified and, in fact, VL K53 is the most modified lysine, occurred in nearly 2/3 of instances. Furthermore, the humanization trials described in WO 2023/168352, demonstrated that any modification to VL K53 negatively impacted the immunoreactivity to nucleic acids of the humanized 3E10-D31N monoclonal antibody (V66). This is further elucidated in Example 6, which show greater uptake of 3E10-D31N monoclonal antibody (V66) oligonucleotide conjugates utilizing transglutaminase-mediated enzymatic conjugation in A427 cells, and in Example 7, which utilize hindered disulfide linker (SPDMV) conjugate (transglutaminase- SPDMV), and imparted the highest exon-skipping (-15%), representing a greater than 10X improvement over lysine-PEG8 AOC. b. Cysteine Attachment
[0444] In some embodiments of AOCs described herein, the linker moiety is conjugated to the antibody or antigen binding fragment therein through a sulfide linkage at one or more surface-exposed cysteine residues on the antibody or antigen binding fragment thereof. In some embodiments of AOCs described herein, the linker moiety is conjugated to the antibody or antigen binding fragment therein through a thiol side chain of one or more cysteine residues on the antibody or antigen binding fragment thereof. In general, antibodies do not possess free thiols, and all cysteine residues form disulfide bonds. In human IgGl, which is commonly used in modem ADCs, there are 4 interchain and 12 intrachain disulfide bonds. The 4 interchain disulfides, which are generally not critical for structural stability of IgGl, can be selectively reduced under mild conditions to give 2, 4, 6, or 8 free thiols while keeping the 12 intrachain disulfides intact. Due to the limited number of conjugation sites and the distinct reactivity of the thiol group, cysteine-based conjugation allows for controlled DAR and heterogeneity. Engineered Cys residues can also be used for site specific conjugation without the partial reduction of the endogenous disulfide bonds using, e.g., EnCys-mAb technology. Many different chemistries are known in the art for attaching payloads to proteins at cystine groups. For example, 8 nucleophilic cysteine residues can first be liberated from the reduced inter-chain disulfide bonds via reducing agents and later conjugated with drug-linker complexes. This approach generates ADCs with heterogeneous conjugation sites and a different number of drugs attached, resulting in a drug to antibody ratio (DAR) ranging from 0~8. Alternatively, partial reduction with either dithiothreitol (DTT) or tri s(2-carboxy ethyl) phosphine (TCEP) can be used to result in the disruption of the heavy-light inter-chain disulfides to release free Cys for drug conjugation, while treatment using 5,5 ’ -dithiobis (2 -nitrobenzoic acid) (DTNB) yields drug conjugates connect to the Cys residues usually involved in heavy-heavy inter- chain disulfides. Further description of cysteine conjugation techniques are described, for example, in Behrens, C.R.; et al., “Methods for site-specific drug conjugation to antibodies,” mAbs. 2014, 6 (1): 46-53; Dennler, P.; et al., “Antibody conjugates: from heterogeneous populations to defined reagents,” Antibodies. 2015, 4: 197-224; and Agarwal, P.; et al., “Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development,” Bioconjugate Chem. 2015, 26: 176-192, the disclosure of which are incorporated herein by reference, in their entireties, for all purposes.
[0445] Accordingly, in some embodiments, the present disclosure provides pharmaceutical compositions comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L- Pr)q, where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein, L is a linker as described herein, P is an oligonucleotide moiety as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, in which L is conjugated to A through cysteine moi eties.
[0446] Advantageously, because of the limited number of conjugation sites and the distinct reactivity of the thiol group, cysteine-based conjugation can be used to generate AOC molecules with a controlled DAR and heterogeneity.
[0447] Accordingly, in some embodiments, a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein will have an average DAR of at least 2. In some embodiments, such a composition will have an average DAR of at least 3. In some embodiments, such a composition will have an average DAR of at least 4. In some embodiments, such a composition will have an average DAR of at least 5. In some embodiments, such a composition will have an average DAR of at least 6. In some embodiments, such a composition will have an average DAR of at least 7. In some embodiments, such a composition will have an average DAR of about 8. In some embodiments, such a composition will have an average DAR of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or about 8.
[0448] Accordingly, in some embodiments, a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 1 and q is at least 2. In some embodiments, r is 1 and q is at least 3. In some embodiments, r is 1 and q is at least 4. In some embodiments, r is 1 and q is at least 5. In some embodiments, r is 1 and q is at least 6. In some embodiments, r is 1 and q is at least 7. In some embodiments, r is 1 and q is about 8. In some embodiments, r is 1 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or 8. In some embodiments, a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 2 (e.g., the linker is a branched linker) and q is at least 2. In some embodiments, r is 2 and q is at least 3. In some embodiments, r is 2 and q is at least 4. In some embodiments, r is 2 and q is at least 5. In some embodiments, r is 2 and q is at least 6. In some embodiments, r is 2 and q is at least 7. In some embodiments, r is 2 and q is about 8. In some embodiments, r is 2 and q is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or about 8. In yet other embodiments, a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, the linker is highly branched, e.g., r is at least 3. In some embodiments, r is at least 4. In some embodiments, r is at least 5, 6, 7, 8, or more. c. Transglutaminase-based Attachment
[0449] In some embodiments of AOCs described herein, the linker moiety is conjugated to the antibody or antigen binding fragment therein through the primary amide side chain of one or more glutamine residues on the antibody or antigen binding fragment thereof. Generally, transglutaminase derived from Streptomyces mobaraensis catalyzes transpeptidation where a primary amine-containing linker is covalently attached to the primary amide side chain of a specific glutamine (Q295) within deglycosylated antibodies, resulting in ADCs with a defined DAR of 2 (one conjugation site per heavy chain) (Jeger et al., “Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase,” Angew Chem Int Ed Engl 2010 49:9995-9997; and Dennler et al., “Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates,” Bioconjugate Chem 2014 25:569-578 the disclosure of which are incorporated herein by reference, in their entireties, for all purposes). An N297Q mutation prior to this conjugation provides two more reaction sites (DAR = 4). This method is quite advantageous in terms of practical ADC production as the glycosidase and transglutaminase directly modify and conjugate native mAbs with the payload, without the need for genetic engineering. An alternative version using a peptide sequence-specific transglutaminase can also be used (Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chem Biol 2013 20: 161-167). This enzyme recognizes and utilizes LLQG (SEQ ID NO: 1037) motif that is genetically incorporated, resulting in site-specific antibody-drug conjugation.
[0450] Accordingly, in some embodiments, the present disclosure provides pharmaceutical compositions comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L- P r)q, where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein, L is a linker as described herein, P is a payload moiety as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16, in which L is conjugated to A through glutamine moieties.
[0451] Accordingly, in some embodiments, a composition comprising an AOC where glutamine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein will have an average Drug Antibody Ratio (DAR) of about 2. In some embodiments, such a composition will have an average DAR of at least 2. In some embodiments, such a composition will have an average DAR of at least 3. In some embodiments, such a composition will have an average DAR of about 4. In some embodiments, such a composition will have an average DAR of about 2, at least 2, at least 3, or about 4.
[0452] Accordingly, in some embodiments, a composition comprising an AOC where glutamine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 1 and q is about 2. In some embodiments, r is 1 and q is at least 2. In some embodiments, r is 1 and q is at least 3. In some embodiments, r is 1 and q is about 4. In some embodiments, r is 1 and q is about 2, at least 2, at least 3, or about 4. In some embodiments, a composition comprising an AOC where glutamine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, r is 2 (e.g., the linker is a branched linker) and q is about 2. In some embodiments, r is 2 and q is at least 2. In some embodiments, r is 2 and q is at least 3. In some embodiments, r is 2 and q is about 4. In some embodiments, r is 2 and q is about 2, at least 2, at least 3, or about 4. In yet other embodiments, a composition comprising an AOC where cysteine attachment is used to conjugate the drug to the antibody or antigen binding fragment thereof as described herein, the linker is highly branched, e.g., r is at least 3. In some embodiments, r is at least 4. In some embodiments, r is at least 5, 6, 7, 8, or more.
2. Cleavable Linkers
[0453] In some aspects, the linker is a cleavable linker. As used herein, “cleavable linker” refers to a linker which can connect two or more molecules and then be cleaved once exposed to an agent. Cleavable linkers can include chemically or enzymatically unstable or degradable linkages. Cleavable linkers generally rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers generally incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker is non-cleavable. In some aspects, the cleavable linker is an acid-labile linker, a phosphatase linker, glucuronidase linker, a cathepsin-B cleavable linker, a cathepsin-L cleavable linker, a protease-sensitive linker, a photo-labile linker, or a disulfide (SPDMV) transglutaminase- containing linker. In some aspects, the cleavable linker is selected from the group consisting of succinyl, O-succinyl, 4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)toluene, sulfosuccinimidyl 6-(3’-(2-pyridyldithio)propionamido)hexanoate, N-succinimidyl-3-(-2-pyridyldithio)- proprionate, succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate, 3-(2-pyridyldithio)- propionyl hydrazide, S-(2-thiopyridyl)-L-cysteine, N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (CAS: 115088-06-7), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) (CAS: 341498-08-6), CL2A, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB), and N- succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP). In some aspects, the cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain. In some embodiments, the linker length of the polyethylene glycol (PEG) chain is 4 PEG molecules, i.e., PEG4. In some embodiments, the PEG chain is PEG8. In some other embodiments, the PEG chain is PEG12. In some aspects, the cleavable homo-bi-functional linker is selected from the group consisting of DSP (Lomant’s Reagent) (CAS: 57757-57-0) and Acid-PEG4-S-S-PEG4- Acid (CAS: 2055015-40-0).
[0454] Additional examples of linkers which contain cleavable disulfide bonds include, but are not limited to “DPDPB”, l,4-di-[3’-(2’-pyridyldithio) propionamido]butane; “SADP”, (N- succinimidyl (4-azidophenyl) l,3’-dithio propionate); “Sulfo-SADP” (Sulfosuccinimidyl (4-azi- dophenyldithio) propionate; “DSP”-Dithio bis (succinimidylproprionate); “DTSSP”-3,3’-Dithio bis (sulfosuccinimidylpropionate); “DTBP” -dimethyl 3,3dithiobispropionimidate-2HCI. Examples of linkers cleavable by oxidation include “DST’-disuccinimidyl tartarate; and “Sulfa- DST”-disuccin imidyl tartarate.
[0455] In some embodiments, the linker L is selected from:
a. Cathepsin-cleavable linkers
[0457] In some embodiments of AOCs described herein, the linker moiety is conjugated to the antibody or antigen binding fragment therein through a cathepsin-cleavable linker.
[0458] AOCs can enter cells via receptor-mediated endocytosis, during intracellular transit or trafficking, and thus they may ultimately encounter the acidic degradative environment of the lysosome which contains multiple proteases and other catalytic enzymes for breakdown of internalized biologic substances. Most often, cleavable linkers therefore leverage an attribute of the lysosome for payload release such as lability to low pH environment, disulfide reducing environment, or cleavage by a lysosomal protease such as cathepsin B. Protease cleavable linkers or (peptide linkers) typically contain a dipeptide sequence based on deduced canonical cleavage specificity for a given protease. Common dipeptide sequences are Valine-Citrulline or Valine- Alanine. These linkers have been used frequently for conjugation of both ADCs and AOCs. [0459] Upon release via linker cleavage, the payloads then require escape from the endosome to traffic to the specific subcellular region in order to mediate an effect.
[0460] 3E10 and its derivatives, including the humanized V66 IgGlK, are anti-DNA antibodies with the unique property of direct cell entry and subsequent trafficking to the nucleus of cells. This occurs via interaction of antibody:DNA complexes with the cell surface transporter ENT2 (equilibrative nucleoside transporter-2) and so does not require the classical endocytic mechanism employing clathrin or dynamin mediated internalization. Further, upon internalization, the antibody does not encounter early or late endosomes and, importantly, does not encounter the harsh environment of the lysosome. As a result, trafficking occurs directly to the nucleus. As such, 3E10 and its derivatives can serve as delivery vehicles for therapeutic payloads that are mechanistically active within the nucleus of target cells.
[0461] Unlike conventional antibody-drug conjugates, it is believed that 3E10 is not transported into the cell through endocytosis and, as such, 3E10 AOCs may not be exposed to the low endosomal pH conditions relied upon in some antibody-drug conjugates for cleavage of pH- sensitive linkers. As such, other strategies are needed for releasing conjugated oligonucleotides from 3E10. In embodiments, an AOC described herein comprises a cleavable linker for which the catalytic agent is present within the within the nucleus for selective release within that organelle.
[0462] It has been found that the cysteine protease Cathepsin L can also be present within the nucleus of cells. See, e.g., Goulet et al., “Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation,” Mol Cancer Res. 2007 Sep;5(9):899-907, incorporated herein by reference in its entirety. This ubiquitous protease has previously been shown to be present primarily in lysosomes as well as in secreted form. Translation initiation within the cathepsin L mRNA for both mouse and human cells has been shown to take place at alternative internal start sites resulting in a polypeptide lacking an NH2 -terminal signal peptide. Other cathepsins typically identified in lysosomes have also been identified in the nucleus, including cathepsin D, cathepsin V, and cathepsin B.
[0463] A nuclear form of the cysteine protease cathepsin-L is found in mouse tumor cells (and also in human tumor cells) (Goulet et al., “Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation,” Mol Cancer Res. 2007 Sep;5(9):899-907; and Soond et al., “Lost or Forgotten: The nuclear cathepsin protein isoforms in cancer,” Cancer Lett. 2019 Oct 10;462:43-50, each incorporated herein by reference in their entireties. Indeed there have been identified several proteases identified that localize to the nucleus but cathepsin-L is most prominent. Further, in cancer cells cathepsin-L appears to play a significant role in cell cycle progression and importantly tumor progression and metastasis.
[0464] In some embodiments, AOCs disclosed herein comprise dipeptides that are substrates for proteolytic cleavage by nuclear localized cathepsins, optionally cathepsin L, cathepsin S, cathepsin D, cathepsin V, and/or cathepsin B.
[0465] In some embodiments, the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-P r)q, where: A is a 3E 10 antibody or antigen-binding fragment thereof as described herein (e.g., as described in the section titled 3E10 Antibodies and Antigen-Binding Fragments), L is a cathepsin-cleavable linker, P is a payload moiety, r is an integer from 1 to 4 and q is an integer from 1 to 16. In some embodiments, L is a cathepsin L- cleavable linker. In some embodiments, L is a cathepsin S-cleavable linker. In some embodiments, L is a cathepsin D-cleavable linker. In some embodiments, L is a cathepsin B-cleavable linker. In some embodiments, L is a cathepsin V-cleavable linker.
[0466] In embodiments, L comprises a dipeptide selected from Phe-Gln, Val-Gln, Leu- Gin, Tyr-Met, Phe-Arg, Phe-Gly, Trp-Thr, Tyr-Gly, Phe-Thr, Val-Gly, Val-Cit, Val-Arg, Thr-Thr, and Val-Thr. In embodiments, L further comprises one or more PEG molecules.
3. Non-Cleavable Linkers
[0467] In some aspects, the linker is a non-cleavable linker. As used herein, “non- cleavable linker” refers to linkers where the release of the biologically active molecule does not depend on, for example, the differential properties between the plasma and some cytoplasmic compartments, or whether the linker has a physical property that permits enzymatic cleavage or chemical cleavage. Non-cleavable linkers can be alkylene chains, or can be polymeric in nature, such as, for example, those based upon polyalkylene glycol polymers, amide polymers, or can include segments of alkylene chains, polyalkylene glycols and/or amide polymers. In some aspects, the non-cleavable linker is selected from DBCO-C6-NHS ester (CAS: 1384870-47-6) and DBCO-PEG8-NHS ester. In some aspects, the non-cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain. In some aspects, the non- cleavable homo-bi-functional linker is selected from DSS (CAS: 68528-80-3) and Bis-PEG8-NHS ester. In embodiments, non-cleavable linkers can include N-succinimidyl (4-iodoacetyl)- aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate, dichlorotriazinic acid, N- succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide), N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), or N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC).
[0468] Additional examples of non-cleavable linkers are “Sulfo-LC-SMPT”- (sulfosuccinimidyl 6-[alphamethyl-alpha-(2-pyridylthio) toluamido}hexanoate; “SMPT” - 4- succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)toluene; “ABH”-Azidobenzoyl hydrazide; “NHS-ASA”-N-Hydroxysuccinimidyl-4-azi dosalicyclic acid; “SASD”- Sulfosuccinimidyl 2-(pazidosali- cylamido)ethyl-l,3-dithiopropionate; “APDP”-N-{4-(p-azi dosalicylamido) buthy}-3’ (2’-pyidyldithio)propionamide; “BASED”-Bis-[ -(4- azidosalicylamido)ethyl]disulfide; “HSAB”-N-hydroxysuccinimidyl-4 azidobenzoate; “APG”- p- Azidophenyl glyoxal monohydrate; “SANPAH”-N-Suc- ciminidyl-6 (4’-azido-2’-mitrophenyl- amimo) hexanoate; “Sulfo-SANPAH”-Sulfosuccinimidyl6-(4’-azido-2’-nitro phenylamino) hexanoate; “ANB-NOS” N-5-Azido-2-ni trobenzyoyloxysuccinimide; “SAND”- Sulfosuccinimidyl-2- (m-azido-o-mitrobenzamido)-ethyl- 1,3’ -dithiopr-opionate; “PNP-DTP”-p- nitrophenyl-2-diazo-3,3,3-trifluoropropi- onate; “SMCC”-Succinimidyl-4-(N- maleimidomethyl)cy clohexane-l-carboxylate; “Sulfo-SMCC”-Sulfosuccinim idyl-4-(N- maleimidomethyl)cyclohexane-l-carboxy-late; “MBS” m-Maleimidobenzoyl-N- hydroxy succinimide ester; “sulfo-MBS”-m-Maleimidobenzoyl-N-hydroxysulfosuccin imide ester; “SIAB”-N-Succinimidyl (4-iodoacetyl)ami nobenzoate; “SulfSIAB”-N-Sulfosuccinimidyl (4-io doacetyl)aminobenzoate; “SMPB”-Succinimidyl 4-(pmalenimidophenyl) butyrate; “Sulfo- SMPB”-Sulfosuc- cinimidyl 4-(p-malenimidophenyl) butyrate; “DSS”-Disuc cinimidyl suberate; “BMH”-Bis maleimidohexane; “BSSS”-bis(sulfosuccinimidyl) suber ate; “DFDNB”-1 ,5- difluoro- 2,4-dinitrobenzene; “DMA”-dimethyl adipimidate 2HCI; “DMP” -Dimethyl pimelimidate-2HCI; “DMS”-dimethyl suberimidate-2-HCI; “SPDPN-succinimidyl-3-(2-py ridylthio) propionate; “Sulfo-HSAB”-Sulfosuccinimidyl 4-(pazidophenyl) butyrate; “Sulfo- SAPB”-Sulfosuccinim idyl 4-(p-azidophenylbutyrate); “ASIB”-l-9p-azidosalicyla mido)-4- (iodoacetamido) butane; “ASBA”-4-(p-Azidosali- cylamido) butylamine.
[0469] Bifunctional chemical linkers include 4-succinimidyl-oxycarbonyl-C-(2- pyridyldithio) toluene; sulfosuccinimidyl-6-O-methyl-O-(pyridyldithiol)-toluamidohe-xanoate; N-succinimidyl-3(2-pyridyldithio)-propri onate; succinimidyl -6-3(-(-2-pyridyldithio)- proprionamidohexanoate; sulfosuccinimidyl-6-3 (-(-2-pyridyldithio)-propionamido hexanoate; 3- (2-pyridyldithio)-propionyl hydrazide, Ellman’s reagent, dichlorotriazinic acid, S-(2-thiopyridyl)- Lcysteine, and the like. Further bifunctional linking molecules are disclosed in U.S. Pat. Nos. 5,349,066; 5,618,528; 4,569,789; 4,952,394; and 5,137,877, each of which is incorporatedherein by reference in its entirety.
[0470] There are a large number of chemical cross-linking agents that are known to those skilled in the art and useful for cross-linking portions of a conjugate. For example, the cross-linking agents are heterobifunctional cross-linkers, which can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers provide the ability to design more specific coupling methods for conjugating, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers.
[0471] A wide variety of heterobifunctional cross-linkers are known in the art, including succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC), m-Maleimidobenzoyl N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB); succinimidyl 4-(p-ma-30 leimidophenyl) butyrate (SMPB); l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)- tolune (SMPT); N-succinimidyl 3-(2-pyridyldithio) propi onate (SPDP); succinimidyl 6-[3-(2- pyridyldithio) propi-onate]hexanoate (LC-SPDP). Those cross-linking agents having N- hydroxysuccinimide moieties can be obtained as the N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional cross-linkers, there exists a number of other cross-linking agents including homobifunctional and photoreactive cross- linkers. Disuccinimidyl subcrate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate 2 HC1 (DMP) are examples of useful homobifunctional cross linking agents, and bis-[B-(4- azidosalicylamido)ethyl]disul fide (BASED) and N-succinimidyl-6(4’-azido-2’-nitrophe nylamino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers.
[0472] One class of heterobifunctional cross-linkers, included above, contain the primary amine reactive group, N-hydroxysuccinimide (NHS), or its water soluble analog N- hydroxysulfosuccinimide (sulfa-NHS). Primary amines (lysine epsilon groups) at alkaline pH’s are unprotonated and react by nucleophilic attack on NHS or sulfa-NHS esters. This reaction results in the formation of an amide bond, and release of NHS or sulfa-NHS as a byproduct. Another reactive group useful as part of a hetero bifunctional cross-linker is a thiol reactive group. Common thiol reactive groups include maleimides, halogens, and pyridyl disulfides. Maleimides react specifically with free sulfhydryls (cysteine residues) in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions. Halogens (iodo acetyl functions) react with -SH groups at physiological pH’s. Both of these reactive groups result in the formation of stable thioether bonds.
[0473] In embodiments, the universal antibody-oligonucleotide conjugate (AOC) provided herein comprises a linker attachment schema selected from the group consisting of: conjugation to exposed lysines, cysteine conjugation following modest reduction of 3E10, engineering free cysteines (e.g., at the C -terminus), site-specific transglutaminase linkage, wherein the linkage can further comprise click chemistry, and any combination thereof. In embodiments, an AOC provided herein further comprises a linker attachment schema comprising engineering free cysteines (e.g., at the C -terminus). In embodiments, an AOC provided herein, comprises an antibody that is cysteine-engineered at the site of linker attachment. In embodiments, an AOC provided herein, comprises a cleavable linker comprising a cleavable disulfide bond. In embodiments, an AOC provided herein comprises a linker wherein the linker is a hindered linker, wherein the linker comprises a carbon atom bearing a sulfur capable of forming a disulfide bond, and wherein the carbon atom is substituted with at least one substituent other than H. In embodiments, the substituent comprises a hydrocarbyl or a substituted hydrocarbyl moiety.
[0474] In some embodiments, the linker L is selected from:
C. Antisense Oligonucleotides [0476] In embodiments, the present disclosure provides an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is an ASO described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16. In embodiments, P is a therapeutic oligonucleotide.
[0477] As used herein, the term “therapeutic oligonucleotide” refers to an oligonucleotide that has a biological, a cytotoxic, or a therapeutic effect in a cell. In embodiments, the therapeutic oligonucleotide is a functional nucleic acid, such as an oligonucleotide or a polynucleotide.
[0478] In embodiments, P is an ASO that mediates exon skipping. In embodiments, an ASO that mediates exon skipping is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site of a pre-mRNA transcript. In embodiments, an ASO that mediates exon skipping is a single stranded oligonucleotide capable of hybridizing to a donor splice site of a pre- mRNA transcript. In embodiments, an ASO that mediates exon skipping is a single stranded oligonucleotide capable of hybridizing to an exonic splice enhancer element of a pre-mRNA transcript. In embodiments, an ASO that mediates exon skipping induces exon skipping in the pre- mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0479] Examples of ASOs that can be used in exon skipping methodologies are shown in Tables 1-3.
[0480] To identify and select antisense oligonucleotides suitable for use in the modulation of exon skipping, a nucleic acid sequence whose function is to be modulated is first identified. This may be, for example, a gene (or mRNA transcribed form the gene) whose expression is associated with a particular disorder or disease state, e.g., Duchenne muscular dystrophy. In embodiments, target site(s) are those involved in mRNA splicing (i.e., splice donor sites, splice acceptor sites, or exonic splicing enhancer elements). In embodiments, splicing branch points and exon recognition sequences or splice enhancers are potential target sites for modulation of mRNA splicing. In embodiments, provided herein are antisense oligonucleotides capable of binding to a selected target in the pre-mRNA to induce efficient and consistent exon skipping.
[0481] The antisense oligonucleotides and pre-mRNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, the term “complementary” is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense molecule need not be 100% complementary to that of its target sequence to interfere with the normal function of the target DNA or RNA as well as to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment.
[0482] The length of an antisense oligonucleotide may vary so long as it is capable of binding selectively to the intended location within the pre-mRNA molecule. The length of such sequences can be determined in accordance with selection procedures described herein. Generally, the antisense oligomer will be from about 10 nucleotides in length up to about 50 nucleotides in length. It will be appreciated however that any length of nucleotides within this range may be used in the method. Preferably, the length of the antisense molecule is between 17 to 30 nucleotides in length.
[0483] To avoid degradation of pre-mRNA during duplex formation with the antisense oligomers, the antisense oligomers used in the method may be adapted to minimize or prevent cleavage by endogenous RNase H. This property is highly preferred as the treatment of the RNA with the unmethylated oligonucleotides either intracellularly or in crude extracts that contain RNase H leads to degradation of the pre-mRNA: antisense oligomer duplexes. Any form of modified antisense molecules that is capable of bypassing or not inducing such degradation may be used in the present method. An example of antisense oligomer which when duplexed with RNA are not cleaved by cellular RNase H is 2'-O-methyl derivatives. 2'-O-methyl-oligoribonucleotides are very stable in a cellular environment and in animal tissues, and their duplexes with RNA have higher Tm values than their ribo- or deoxyribo-counterparts.
[0484] Antisense oligonucleotides that do not activate RNase H can be made in accordance with known techniques, see, e.g., U.S. Pat. No. 5, 149,797. Such antisense oligonucleotides, which may be deoxyribonucleotide or ribonucleotide sequences, simply contain any structural modification which sterically hinders or prevents binding of RNase H to a duplex molecule containing the oligonucleotide as one member thereof, which structural modification does not substantially hinder or disrupt duplex formation. Because the portions of the oligonucleotide involved in duplex formation are substantially different from those portions involved in RNase H binding thereto, numerous antisense molecules that do not activate RNase H are available. For example, such antisense oligonucleotides wherein at least one, or all, of the inter-nucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphorothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues may be modified as described. In another non-limiting example, such antisense oligonucleotides are oligonucleotides wherein at least one, or all, of the nucleotides contain a 2' lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2- propenyl, and isopropyl). For example, every other one of the nucleotides may be modified as described.
[0485] The present disclosure appreciates other antisense oligonucleotides including, but not limited to, oligonucleotide mimetics.
[0486] Specific examples of preferred antisense compounds useful in this disclosure include oligonucleotides containing modified backbones or non-natural inter-nucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, and referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their inter-nucleoside backbone can also be considered to be oligonucleosides.
[0487] In some embodiments, the oligonucleotide mimetics, both the sugar and the inter- nucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
[0488] Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligomers obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993). The backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.
[0489] Modified oligonucleotides may also contain one or more substituted sugar moi eties. Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Certain nucleo-bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
[0490] Another modification of the oligonucleotides of the disclosure involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di- hexadecyl-rac -glycerol or tri ethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
[0491] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present disclosure also includes antisense that are chimeric compounds.
[0492] Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid. /. Example ASOs for Exon Skipping
[0493] In some embodiments, the exon-skipping inducing oligonucleotides have a sequence selected from SEQ ID NO:99-103, as detailed in Figure 15, or any variation thereof disclosed herein. In other embodiments, the exon-skipping inducing oligonucleotides can be selected from the group consisting of SEQ ID NO: 150-398, as detailed in Table 2. In some embodiments, the exon-skipping inducing oligonucleotides can be selected from the group consisting of SEQ ID NO:410-988, as detailed in Table 3. It should be understood that the oligonucleotides disclosed in Table 2 and Table 3 are exemplary in nature, and in no way limiting the present invention.
Table 1 - Example genes targeted by exon skipping oligos.
Table 2 - Example sequences of oligos for use in treating various disorders.
Table 3 - Example single stranded oligonucleotide sequences capable of inducing exon skipping
[0494]
I). Exemplary Linker-Antibodies
[0495] In embodiments, the present disclosure provides an antibody-payload conjugate having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16.
[0496] In some embodiments, L is a cathepsin-L cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0497] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0498] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0499] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0500] In some embodiments, L is a cathepsin-L cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH
CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gln- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide, In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0501] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0502] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0503] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0504] In some embodiments, L is a cathepsin-L cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Lhr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0505] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0506] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0507] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0508] In some embodiments, L is a cathepsin-L cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val- Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp- Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0509] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0510] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0511] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0512] In some embodiments, is a cathepsin-L cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val- Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gln- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Met- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Arg- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gly- dipeptide. In some embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp- Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Tyr-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Thr- dipeptide. In some embodiments, the cathepsin-L cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0513] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0514] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0515] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker and an antibody or antigen- binding fragment thereof that comprises Heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0516] In some embodiments, L is a cleavable linker comprising a -Phe-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0517] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms. [0518] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0519] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0520] In some embodiments, L is a cleavable linker comprising a -Phe-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0521] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms. [0522] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0523] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0524] In some embodiments, L is a cleavable linker comprising a -Phe-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0525] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms. [0526] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0527] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 1 , 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0528] In some embodiments, L is a cleavable linker comprising a -Phe-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0529] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90, the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0530] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0531] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0532] In some embodiments, L is a cleavable linker comprising a -Phe-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0533] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the cleavable linker comprising a -Phe-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Phe-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0534] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 1 17 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
4 to 10.
[0535] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Phe-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0536] In some embodiments, L is a cleavable linker comprising a -Val-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0537] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0538] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10. [0539] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0540] In some embodiments, L is a cleavable linker comprising a -Val-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. Tn some embodiments, the cleavable linker comprising a -Vai -Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0541] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0542] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0543] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0544] In some embodiments, L is a cleavable linker comprising a -Val-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0545] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0546] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0547] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0548] In some embodiments, L is a cleavable linker comprising a -Val-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0549] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO, the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0550] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0551] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0552] In some embodiments, L is a cleavable linker comprising a -Val-Gln- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 1 17. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0553] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the cleavable linker comprising a -Val-Gln- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Val-Gln- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0554] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided. Tn some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0555] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Val-Gln- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein. [0556] In some embodiments, L is a cleavable linker comprising a -Leu-Gin- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0557] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0558] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. Tn some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0559] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0560] In some embodiments, L is a cleavable linker comprising a -Leu-Gin- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NOVO. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0561] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0562] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0563] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0564] In some embodiments, L is a cleavable linker comprising a -Leu-Gin- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0565] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0566] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0567] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0568] In some embodiments, L is a cleavable linker comprising a -Leu-Gin- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0569] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90, the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from
3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0570] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
4 to 10. [0571] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0572] In some embodiments, L is a cleavable linker comprising a -Leu-Gin- dipeptide, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0573] In some embodiments of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the cleavable linker comprising a -Leu-Gin- dipeptide is an un-branched linker. In some embodiments, the cleavable linker comprising a -Leu-Gin- dipeptide is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0574] In some embodiments, compositions of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 1 17 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0575] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cleavable linker comprising a -Leu-Gin- dipeptide and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0576] In some embodiments, L is a disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0577] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the disulfide cleavable linker is an un-branched linker. In some embodiments, the disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. Tn some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0578] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0579] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0580] In some embodiments, L is a disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0581] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the disulfide cleavable linker is an un-branched linker. In some embodiments, the disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0582] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0583] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0584] In some embodiments, L is a disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 17. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0585] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the disulfide cleavable linker is an un-branched linker. In some embodiments, the disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms. [0586] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0587] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0588] In some embodiments, L is a disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0589] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, the disulfide cleavable linker is an un-branched linker. In some embodiments, the disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0590] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0591] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0592] In some embodiments, L is a disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue. [0593] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the disulfide cleavable linker is an un- branched linker. In some embodiments, the disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0594] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0595] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 1 17, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0596] In some embodiments, L is a hindered disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NOVO. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen- binding fragment thereof at a lysine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0597] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61 , 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the hindered disulfide cleavable linker is an un-branched linker. In some embodiments, the hindered disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0598] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0599] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0600] In some embodiments, L is a hindered disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen- binding fragment thereof at a lysine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0601] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the hindered disulfide cleavable linker is an un-branched linker. In some embodiments, the hindered disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0602] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0603] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0604] In some embodiments, L is a hindered disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0605] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the hindered disulfide cleavable linker is an un-branched linker. In some embodiments, the hindered disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0606] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0607] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0608] In some embodiments, L is a hindered disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen- binding fragment thereof at a cysteine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0609] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, the hindered disulfide cleavable linker is an un-branched linker. In some embodiments, the hindered disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0610] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0611] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen- binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0612] In some embodiments, L is a hindered disulfide cleavable linker, and A is an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen- binding fragment thereof at a cysteine residue. In some embodiments, the hindered disulfide cleavable linker is attached to the antibody or antigen-binding fragment thereof at a glutamine residue.
[0613] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the hindered disulfide cleavable linker is an un-branched linker. In some embodiments, the hindered disulfide cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0614] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen-binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
4 to 10.
[0615] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody -payload conjugate with a hindered disulfide cleavable linker and an antibody or antigen- binding fragment thereof that comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
F. Exemplary Conjugation Chemistry-Linkers
[0616] In embodiments, the present disclosure provides an antibody-payload conjugates having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16.
[0617] In embodiments, L is a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide. In embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide. In embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Tyr-Met- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Arg- dipeptide. Tn embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Gly- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Tyr-Gly- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Thr- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:117.
[0618] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0619] In some embodiments, compositions of an antibody -pay load conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0620] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0621] In embodiments, L is a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the cathepsin-L cleavable linker comprises a -Phe-Gln- dipeptide. In embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide. In embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Tyr-Met- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Arg- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Gly- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Tyr-Gly- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Thr- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:117.
[0622] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0623] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0624] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0625] In some embodiments, the present disclosure provides pharmaceutical compositions comprising an antibody -payload conjugate having the formula A-(L-Pr)q, where: A is a 3E10 antibody or antigen-binding fragment thereof as described herein (e g., as described in the section titled 3E10 Antibodies and Antigen-Binding Fragments), L is a linker, P is a payload as described herein, r is an integer from 1 to 4 and q is an integer from 1 to 16.
[0626] In embodiments, L is a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the cathepsin- L cleavable linker comprises a -Phe-Gln- dipeptide. In embodiments, the cathepsin-L cleavable linker comprises a -Val-Gln- dipeptide. In embodiments, the cathepsin-L cleavable linker comprises a -Leu-Gin- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Tyr-Met- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Arg- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Gly- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Trp-Thr- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Tyr-Gly- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Phe-Thr- dipeptide. In embodiments, the cathepsin-L comprises cleavable linker comprises a -Val-Gly- dipeptide. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:117.
[0627] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0628] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0629] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody -payload conjugate with a cathepsin-L cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0630] In embodiments, L is a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0631] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a lysine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0632] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0633] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0634] In embodiments, L is a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0635] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a cysteine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0636] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0637] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, as described above. Tn some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon- skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0638] In embodiments, L is a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0639] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a glutamine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0640] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0641] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Phe-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon- skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0642] In embodiments, L is a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0643] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a lysine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0644] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0645] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0646] In embodiments, L is a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ IDNOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0647] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a cysteine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0648] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0649] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon- skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0650] In embodiments, L is a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. [0651] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a glutamine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0652] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody -payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0653] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Val-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon- skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0654] In embodiments, L is a cathepsin-L cleavable linker comprising a -Leu-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0655] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a lysine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms. [0656] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0657] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein. [0658] In embodiments, L is a cathepsin-L cleavable linker comprising a -Leu-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ IDNOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0659] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a cysteine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0660] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0661] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon- skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0662] In embodiments, L is a cathepsin-L cleavable linker comprising a -Leu-Gln- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen- binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0663] In some embodiments of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen- binding fragment thereof at a glutamine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0664] In some embodiments, compositions of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody -payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0665] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a cathepsin-L cleavable linker comprising a -Leu-Gin- dipeptide attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon- skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0666] In embodiments, L is a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0667] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0668] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0669] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0670] In embodiments, L is a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0671] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0672] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0673] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0674] In embodiments, L is a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0675] In some embodiments of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0676] In some embodiments, compositions of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
4 to 10.
[0677] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0678] In embodiments, L is a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. [0679] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0680] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0681] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a lysine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
[0682] In embodiments, L is a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0683] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms. [0684] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0685] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a cysteine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein. [0686] In embodiments, L is a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen- binding fragment thereof comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NOVO. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117.
[0687] In some embodiments of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, the cathepsin-L cleavable linker is an un-branched linker. In some embodiments, the cathepsin-L cleavable linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0688] In some embodiments, compositions of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody- payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0689] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with a hindered disulfide cleavable linker attached to the 3E10 antibody or antigen-binding fragment thereof at a glutamine residue, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. In some embodiments, the oligonucleotide (P) is an antisense oligonucleotide (ASO). In some embodiments, the ASO mediates exon skipping. In some embodiments, the ASO is a single stranded oligonucleotide capable of hybridizing to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of a pre-mRNA transcript, wherein the single stranded oligonucleotide induces exon skipping in the pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein.
G. Exemplary Antibody-Payload Conjugates
[0690] In embodiments, the present disclosure provides an antibody-payload conjugate having the formula A-(L-Pr)q, where: A is a 3E10 antibody or antigen-binding fragment thereof (e.g., as described in the section titled 3E10 Antibodies and Antigen-Binding Fragments), L is a linker as described herein, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16.
[0691] In some embodiments, P is an ASO that mediates exon skipping, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the linker is a cathepsin-L cleavable linker. In some embodiments, the linker is a disulfide cleavable linker. In some embodiments, the linker is a hindered disulfide linker.
[0692] In some embodiments of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, the linker connecting the antibody or antigen binding fragment thereof to the ASO that mediates exon skipping is an un- branched linker. In some embodiments, the linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0693] In some embodiments, compositions of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0694] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 61, 62, and 63 and VH CDRs of SEQ ID NOs: 58, 59, and 60, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
[0695] In some embodiments, P is an ASO that mediates exon skipping, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5. In some embodiments, the antibody or antigen- binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:69 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO:90. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 69 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:90. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the linker is a cathepsin-L cleavable linker. In some embodiments, the linker is a disulfide cleavable linker. In some embodiments, the linker is a hindered disulfide linker.
[0696] In some embodiments of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, the linker connecting the antibody or antigen binding fragment thereof to the ASO that mediates exon skipping is an un- branched linker. In some embodiments, the linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0697] In some embodiments, compositions of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0698] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 9, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 4, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
[0699] In some embodiments, P is an ASO that mediates exon skipping, and A is an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region (VH) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a VH with an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 105 and a VL with an amino acid sequence having at least 99% sequence identity to SEQ ID NO:117. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a cysteine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the linker is a cathepsin-L cleavable linker. In some embodiments, the linker is a disulfide cleavable linker. In some embodiments, the linker is a hindered disulfide linker.
[0700] In some embodiments of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, the linker connecting the antibody or antigen binding fragment thereof to the ASO that mediates exon skipping is an un- branched linker. In some embodiments, the linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from
3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0701] In some embodiments, compositions of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody- payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody -payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from
4 to 10. [0702] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen- binding fragment thereof that comprises VL CDRs of SEQ ID NOs: 29, 10, and 11 and VH CDRs of SEQ ID NOs: 15, 26, and 5, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
[0703] In some embodiments, P is an ASO that mediates exon skipping, and A is an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen- binding fragment thereof at a cysteine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the linker is a cathepsin-L cleavable linker. In some embodiments, the linker is a disulfide cleavable linker. In some embodiments, the linker is a hindered disulfide linker.
[0704] In some embodiments of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, the linker connecting the antibody or antigen binding fragment thereof to the ASO that mediates exon skipping is an un-branched linker. In some embodiments, the linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0705] In some embodiments, compositions of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO:69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 90 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0706] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen- binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 69 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO:90, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies. [0707] In some embodiments, P is an ASO that mediates exon skipping, and A is an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a lysine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen- binding fragment thereof at a cysteine residue. In some embodiments, the ASO that mediates exon skipping is attached to the antibody or antigen-binding fragment thereof at a glutamine residue. In some embodiments, the linker is a cathepsin-L cleavable linker. In some embodiments, the linker is a disulfide cleavable linker. In some embodiments, the linker is a hindered disulfide linker.
[0708] In some embodiments of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, the linker connecting the antibody or antigen binding fragment thereof to the ASO that mediates exon skipping is an un-branched linker. In some embodiments, the linker is a branched linker. In some embodiments, the branched linker has two arms. In some embodiments, the branched linker has three arms. In some embodiments, the branched linker has four arms. In some embodiments, the branched linker has at least 2 arms, at least 3 arms, at least 4 arms, or more. In some embodiments, the branched linker has from 2 to 6 arms, from 2 to 5 arms, from 2 to 4 arms, from 2 to 3 arms, from 3 to 6 arms, from 3 to 5 arms, from 3 to 4 arms, from 4 to 6 arms, or from 5 to 6 arms.
[0709] In some embodiments, compositions of an antibody-payload conjugate with an ASO that mediates exon skipping and an antibody or antigen-binding fragment thereof that comprises heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117 are provided. In some such compositions, the average drug to antibody ratio (DAR) for antibody-payload conjugates in the composition at least 2. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 4. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 6. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 8. In some such compositions, the average DAR for antibody-payload conjugates in the composition is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or greater. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 2 to 4, from 2 to 5, from 2 to 6, from 2 to 8, or from 2 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 3 to 4, from 3 to 5, from 3 to 6, from 3 to 8, or from 3 to 10. In some embodiments, the average DAR for antibody-payload conjugates in the composition is from 4 to 5, from 4 to 6, from 4 to 8, or from 4 to 10.
[0710] In some embodiments, methods are provided for treating a genetic disease or disorder by administering, to a subject in need thereof, a therapeutically effective amount of an antibody -payload conjugate with an ASO that mediates exon skipping and an antibody or antigen- binding fragment thereof that comprises Heavy chain variable region (VH) with an amino acid sequence of SEQ ID NO: 105 and a light chain variable region (VL) with an amino acid sequence of SEQ ID NO: 117, as described above. In some embodiments, the genetic disease or disorder is a neurogenetic disease. In some embodiments, the genetic disease or disorder is a musculoskeletal disorder. In some embodiments, the genetic disease or disorder is a cardiovascular disease. In some embodiments, the genetic disease or disorder is a metabolic disease. In some embodiments, the genetic disease or disorder is a cancer. In some embodiments, the genetic disease or disorder is a lung disorder. In some embodiments, the genetic disease or disorder is a diseases that can be benefitted by exon-skipping therapies.
IV. Pharmaceutical Compositions
[0711] In one aspect, the present disclosure provides pharmaceutical compositions including a complex formed between a therapeutic oligonucleotide, e.g., as described above, and a 3E10 antibody or antigen-binding fragment thereof, as described herein.
[0712] In embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is an oligonucleotide as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16; wherein the 3E10 antibody or antigen-binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of XI ASX2X3VSTSSYSYX4X5, where XI is K, R, or H, X2 is K, R, or H, X3 is T or S, X4 is M or L, and X5 is K, R, H, or A (SEQ ID NO:61), (b) a VL CDR2 comprising the amino acid sequence of YASYLX1S, where XI is D, E, N, or Q (SEQ ID NO:62), and (c) a VL CDR3 comprising the amino acid sequence of QX1SX2X3FPWT, where XI is K, R, or H, X2 is K, R, or H, and X3 is D or E (SEQ ID NO:63), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of X1YGMX2, where XI is D, E, N, Q, R, or K and X2 is K, R, or H (SEQ ID NO:58), (e) a VH CDR2 comprising the amino acid sequence of YISSX1SSTIYYAX2X3VX4G, where XI is G or S, X2 is D or E, X3 is T or S, and X4 is K, R, or H (SEQ ID NO:59), and (f) a VH CDR3 comprising the amino acid sequence of X1GLLLX2Y, where XI is K, R, or H, and X2 is D or E (SEQ ID NO:60), and a pharmaceutically acceptable carrier.
[0713] In embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a 3E 10 antibody or antigen-binding fragment thereof, L is a linker, P is an oligonucleotide as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16; wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15), and a pharmaceutically acceptable carrier. In embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of RASKS VSTS SYS YMH (SEQ ID NO:9), (b) a VL CDR2 comprising the amino acid sequence of YASYLES (SEQ ID NO: 10), and (c) a VL CDR3 comprising the amino acid sequence of QHSREFPWT (SEQ ID NO: 11), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15), (e) a VH CDR2 comprising the amino acid sequence of YISSGSSTIYYADTVKG (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of RGLLLDY (SEQ ID NO: 5), and a pharmaceutically acceptable carrier. In embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence that is identical to SEQ ID NO:21. In embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence that is identical to SEQ ID NO: 14. In embodiments, the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence that is identical to SEQ ID NO:20. In embodiments, the antibody or antigen-binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is identical to SEQ ID NO: 13.
[0714] In embodiments of pharmaceutical compositions comprising AOCs provided herein, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with R. In embodiments, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with L. In embodiments, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with K.
[0715] In embodiments of pharmaceutical compositions comprising an AOC provided herein, the antibody or antigen-binding fragment thereof is a murine, a chimeric, a humanized, or a human antibody.
[0716] In embodiments of the pharmaceutical composition comprising an antibody- oligonucleotide conjugate (AOC) provided herein, L is a cleavable linker. In embodiments, L is selected from the succinyl, O-succinyl, 4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)toluene, sulfosuccinimidyl 6-(3’ -(2 -pyridyl dithio)propionamido)hexanoate, N-succinimidyl-3-(-2- pyridyldithioj-proprionate, succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate, 3-(2- pyridyldithioj-propionyl hydrazide, S-(2-thiopyridyl)-L-cysteine, N-succinimidyl 4-(2- pyridyldithiojbutanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo- SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), CL2A, maleimidocaproyl-valine- citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB) and N-succinimidyl 4-(2-pyridyldithio)-2- sulfopentanoate (sulfo-SPP).In embodiments of the pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) provided herein, L is a non-cleavable linker. In embodiments, L is selected from N-succinimidyl (4-iodoacetyl)-aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate, dichlorotriazinic acid, N-succinimidyl-[(N- maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), or N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC).
[0717] In embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a humanized 3E10 antibody or antigen-binding fragment thereof comprising: (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:9), (b) a VL CDR2 comprising the amino acid sequence of YASYLES (SEQ ID NO: 10), and (c) a VL CDR3 comprising the amino acid sequence of QHSREFPWT (SEQ ID NO: 11), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15), (e) a VH CDR2 comprising the amino acid sequence of YISSGSSTIYYADTVKG (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of RGLLLDY (SEQ ID NO: 5), L is a linker described herein, and P is an oligonucleotide as described herein, and a pharmaceutically acceptable carrier.
[0718] In embodiments, the present disclosure provides a pharmaceutical composition comprising an AOC disclosed herein that has the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is an oligonucleotide as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16; wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 16), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain, and wherein D31 is wild-type 3E10 CDR1, and a pharmaceutically acceptable carrier.
[0719] In some aspects of pharmaceutical compositions comprising the antibody- oligonucleotide conjugate (AOC) provided herein, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with R. In some aspects, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with L. In some aspects, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with K.
[0720] In some aspects of the pharmaceutical composition comprising an 3E10 antibody - oligonucleotide conjugate (AOC) provided herein, the 3E10 antibody or antigen-binding fragment thereof is murine, chimeric, humanized, or human.
[0721] In some aspects of the pharmaceutical composition comprising an antibody- oligonucleotide conjugate (AOC) provided herein, comprises a cleavable linker. In some aspects, the cleavable linker is an acid-labile linker, a protease-sensitive linker, a photo-labile linker, or a disulfide-containing linker. In some aspects, the cleavable linker is selected from the group consisting of SPDB (CAS: 115088-06-7) or SPP (CAS: 341498-08-6). In some aspects, the cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain. In some aspects, the cleavable homo-bi-functional linker is selected from the group consisting ofDSP (Lomant’s Reagent) (CAS: 57757-57-0) and Acid-PEG4-S-S-PEG4-Acid (CAS: 2055015-40-0).
[0722] In some aspects of the pharmaceutical composition comprising an antibody- oligonucleotide conjugate (AOC) provided herein, comprises a non-cleavable linker. In some aspects, the non-cleavable linker is selected from DBCO-C6-NHS ester (CAS: 1384870-47-6) and DBCO-PEG8-NHS ester. In some aspects, the non-cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain. In some aspects, the non- cleavable homo-bi-functional linker is selected from DSS (CAS: 68528-80-3) and Bis-PEG8-NHS ester.
[0723] In some aspects, the present disclosure provides a pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) comprising: VH complementarity determining regions (CDRs) having the amino acid sequences ofNYGMH (SEQ ID NO: 15); CDR H2.1 : YISSGSSTIYYADTVKG (SEQ ID NO:4); CDR H3.1: RGLLLDY (SEQ ID NO:5), and VL CDRs having the amino acid sequences of RASKS VSTS SYS YMH (SEQ ID NO:9); CDR L2.1 : YASYLES (SEQ ID NO: 10); CDR L3.1 : QHSREFPWT (SEQ ID NO l l), SPP (CAS: 341498-08-6), and oligonucleotide as described herein.
[0724] Because 3E10 antibodies or variants thereof, or antigen-binding fragments thereof localize to tissues in vivo following systemic administration, the compositions of the present disclosure can be formulated for, and subsequently administered by, one of many common administrative routes. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the parenteral administration is intramuscular administration, intravenous administration, or subcutaneous administration.
A. Dosages
[0725] The term “pharmaceutically acceptable” refers to the fact that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. For example, the carrier, diluent, or excipient or composition thereof can be administered to a subject along with an antibody-drug conjugate (AOC) of the disclosure without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained. Pharmaceutical compositions including the AOCs can be administered by any suitable means, for example, parenterally, such as by subcutaneous, intravenous, intramuscular, intrathecal, or intracistemal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous Solutions or Suspensions) in dosage formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. In some aspects the conjugate is administered parenterally, or intravenously.
[0726] The pharmaceutical compositions of an AOC described herein can be administered either alone or in combination with other therapeutic agents, can conveniently be presented in unit dose form and can be prepared by any of the methods well known in the art of pharmacy. All methods include bringing the conjugate into association with the carrier, which constitutes one or more accessory ingredients, are contemplated. Techniques for formulation and administration are generally known in the art. Suitable routes can be, for example, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, topical, epidermal, or mucosal administration.
V. Methods of Use
[0727] In some aspects, the present disclosure provides methods of treating a disease or disorder, optionally a genetic disease or disorder. The compositions, conjugates, and methods described herein are useful for treating subjects having genetic diseases or disorders by delaying or inhibiting progression and/or symptoms of the disease or disorder.
[0728] In some aspects, the present disclosure provides a method of treating a genetic disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) having the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, optionally wherein L is a cleavable linker, P is an oligonucleotide moiety as described herein, optionally wherein P is an ASO as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16; and wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO:15). In some aspects, the 3E10 antibody or antigen-binding fragment thereof comprises (a) a light chain variable region (VL) complementarity determining region (CDR) 1 comprising the amino acid sequence of RASKSVSTSSYSYMH (SEQ ID NO:9), (b) a VL CDR2 comprising the amino acid sequence of YASYLES (SEQ ID NOTO), and (c) a VL CDR3 comprising the amino acid sequence of QHSREFPWT (SEQ ID NO: 1 1), and (d) a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of NYGMH (SEQ ID NO: 15), (e) a VH CDR2 comprising the amino acid sequence of YISSGSSTIYYADTVKG (SEQ ID NO: 4), and (f) a VH CDR3 comprising the amino acid sequence of RGLLLDY (SEQ ID NO: 5).
[0729] In some aspects, the 3E10 antibody or antigen-binding fragment thereof comprises VH complementarity determining regions (CDRs) having the amino acid sequences of NYGMH (SEQ ID NO: 15); CDR H2.1 : YISSGSSTIYYADTVKG (SEQ ID NO:4); CDR H3.1 : RGLLLDY (SEQ ID NO:5), and VL CDRs having the amino acid sequences of RASKSVSTSSYSYMH (SEQ ID NO:9); CDR L2.1 : YASYLES (SEQ ID NO: 10); CDR L3.1 : QHSREFPWT (SEQ ID NO: 11), and a pharmaceutically acceptable carrier.
[0730] In some aspects, the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having an amino acid sequence that is identical to 3E10- HC D31N (SEQ ID NO: 13) or 3E10-VH D31N (SEQ ID NO: 14); and a light chain variable region (VL) having an amino acid sequence that is identical to 3E10-VL D3 IN (SEQ ID NO:20) or 3E10-VL-VR D31N (SEQ ID NO:21). In embodiments, the antibody or antigen-binding fragment thereof comprises a full length heavy chain (HC) comprising an amino acid sequence that is identical to SEQ ID NO:13.
[0731] In some aspects of the methods disclosed herein, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with R. In some aspects, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with L. In some aspects, the amino acid residue corresponding with N31 of the heavy chain CDR1 is substituted with K.
[0732] In some aspects of the methods disclosed herein, the antibody or antigen-binding fragment thereof is murine, chimeric, humanized, or human.
[0733] In some aspects of the methods disclosed herein, the pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) provided herein, is a cleavable linker. In some aspects, (L) the cleavable linker is an acid-labile linker, a protease-sensitive linker, a photo-labile linker, or a disulfide-containing linker. In some aspects, the cleavable linker is selected from the group consisting of SPDB (CAS: 115088-06-7) or SPP (CAS: 341498-08-6). In some aspects, the cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain. In some aspects, the cleavable homo-bi-functional linker is selected from the group consisting of DSP (Lomant’s Reagent) (CAS: 57757-57-0) and Acid- PEG4-S-S-PEG4-Acid (CAS: 2055015-40-0).
[0734] In some aspects of the methods disclosed herein, the pharmaceutical composition comprising an antibody-oligonucleotide conjugate (AOC) provided herein, is a non-cleavable linker. In some aspects, the non-cleavable linker is selected from DBCO-C6-NHS ester (CAS: 1384870-47-6), Az/DBCO-PEG 8 and DBCO-PEG8-NHS ester. In some aspects, the non- cleavable linker is a homo-bi-functional linker, optionally comprising an alkyl or polyethylene glycol (PEG) chain. In some aspects, the non-cleavable homo-bi-functional linker is selected from DSS (CAS: 68528-80-3) and Bis-PEG8-NHS ester.
[0735] In embodiments of the methods disclosed herein, L is a cleavable linker. In embodiments, L is selected from succinyl, O-succinyl, 4-succinimidyl-oxycarbonyl-a-(2- pyridyldithio)toluene, sulfosuccinimidyl 6-(3’-(2-pyridyldithio)propionamido)hexanoate, N- succinimidyl-3-(-2-pyridyldithio)-propri onate, succinimidyl 6-(3(2- pyridyldithio)propionamido)hexanoate, 3-(2-pyridyldithio)-propionyl hydrazide, S-(2- thiopyridyl)-L-cysteine, N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4- (2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), CL2A, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB) and N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfo-SPP).
[0736] In embodiments of the methods disclosed herein, L is a non-cleavable linker. In embodiments, L is selected from N-succinimidyl (4-iodoacetyl)-aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate, dichlorotriazinic acid, N-succinimidyl-[(N- maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-maleimide), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), or N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC).
[0737] In embodiments, the present disclosure provides a method of treating a genetic disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an AOC disclosed herein that has the formula A-(L-Pr)q, wherein: A is a 3E10 antibody or antigen-binding fragment thereof, L is a linker, P is a payload as described herein, r is an integer from 1 to 4, and q is an integer from 1 to 16; wherein the 3E10 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of XYGMH (SEQ ID NO: 16), wherein X = D, N, R, L, or K and corresponds to an amino acid residue at position 31 of the 3E10 heavy chain, and wherein D31 is wild-type 3E10 CDR1.
A. _ Genetic Diseases and Disorders
[0738] The compositions described herein are well suited for the delivery of antisense oligonucleotides useful for treating various genetic diseases. Examples of proteins, and their associated genes, that are mutated in various neurogenetic, cardiovascular, metabolic, cancer, musculoskeletal, and lung diseases are presented in Table 1. Generally, sequences encoding, or complementary to sequences encoding, any one of these proteins, and variants thereof retaining a function of the full-length protein, can be included in the therapeutic polynucleotides disclosed herein. Accordingly, in some embodiments, the polypeptide is selected from the group consisting of ataxia telangiectasia mutated (ATM), phosphomannomutase 2 (PMM2), microtubule-associated protein tau (MAPT), Niemann-Pick Cl (NPC1), Neurofibromin (NF1), Merlin (NF2), Oligomeric plasma membrane (MLC1), Proteolipid protein (PLP1), Inhibitor kinase complex-associated protein (IKBKAP aka ELP1), Survival of motor neuron (SMN2), Dystrophia myotonica protein kinase (DMPK), Cellular nucleic acid-binding protein (CNBP/ZNF9), lagged canonical Notch ligand 1 (JAG1), TSC complex subunit 2 (TSC2), usherin (USH2A), adenosine deaminase RNA specific (ADAR), ATPase Na+/K+ transporting subunit alpha 2 (ATP1 A2), prickle planar cell polarity protein 2 (PRICKLE2), SET domain containing 5 (SETD5), eukaryotic translation ini- tiation factor 2B subunit epsilon (EIF2B5), eukaryotic translation initiation factor 2B alpha (EIF2B1), eukaryotic translation initiation factor 2B subunit beta (EIF2B2), peroxisomal bio- genesis factor 1 (PEX1), syntaxin binding protein 1 (STXBP1), proline rich transmembrane pro- tein 2 (PRRT2), syntaxin IB (STX1B), retinoic acid induced 1 (RAI1), transcription factor 4 (TCF4), calcium voltage-gated channel subunit alpha 1 A (CACNA1A), DNA methyltransferase 1 (DNMT1), SH3 and multiple ankyrin repeat domain 3 (SHANK3), arylsulfatase A (ARSA), Prelamin A (LMNA), dystrophin (DMD), myostatin (MSTN), phenylalanine hydroxylase (PAH), and Apolipoprotein B (APOB). 1. Neurogenetic diseases
[0739] Neurogenetic diseases are typically characterized by mutations that affect the splicing process The brain expresses a relatively higher number of alternatively spliced genes, some of which were found to be linked to several neurological, neuromuscular, and neurodegenerative diseases. Because the etiology of many different forms of neurogenetic diseases have been well characterized, gene therapies offer an attractive option for treating these diseases. In fact, there are approved therapies and ongoing clinical trials for such gene therapies for several neurogenetic diseases (Siva K, Covello G, Denti MA. Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases. Nucleic Acid Ther. 2014 Feb;24(l):69-86).
CDG
[0740] One such disorder for which a gene therapy is being developed is congenital disorder of glycosylation (CDG). CDG is an autosomal recessive disorder that affects glycan synthesis. The most prevalent form of CDG, type la (OMIM 212065), has an incidence of 1 in 50,000 to 1 in 100,000 individuals, and is caused by mutations in PMM2 gene, which is located on chromosome 16pl3 and the gene encodes phosphomannomutase 2 protein (PMM2), a key enzyme that controls the synthesis of GDP -mannose which is essential for the generation of N- glycans. Mutations in the PMM2 gene lead to the hypoglycosylation of different proteins in different tissues (Dupre et al. 2000., Glycobiology 10, 1277-1128). Since the total lack of the PMM2 gene product is lethal, no patient with two copies of any inactivating mutation has ever been recorded. In one study, two different 25-nt-long phosphorodiamidate morpholino oligomers (PMOs) were designed, complementary to the 5’ or 3’ cryptic splice sites of the pseudoexon in intron 7 and both these PMOs were transfected at the same time into patient’s fibroblasts carrying the c.640- 15479C > T mutation in heterozygosity with missense mutationc.691G > A. The results showed that there was a 100% restoration of the correctly spliced mRNA. The levels of PMM2 protein after transfection increased from 9% to 23% of the quantity detected for the control cell line. PMM2 enzymatic activity was rescued almost to 50% that of the control fibroblasts (Vega et al., 2009, Hum. Mutat. 30(5);795-803).
[0741] In some embodiments, the methods and compositions described herein are useful for treating CDG, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in PMM2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, or 8 target regions of the PMM2 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 2 target region of the PMM2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 3 target region of the PMM2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 4 target region of the PMM2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 5 target region of the PMM2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 6 target region of the PMM2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 7 target region of the PMM2 pre-mRNA designated as an annealing site.
[0742] In some embodiments, antisense oligonucleotides of the disclosure target PMM2 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, and/or 8, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, and/or 8, the disrupted reading frame is restored to an in-frame mutation. While CDG is comprised of various genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 1, 2, 3, 4, 5, 6, 7, and/or 8, of PMM2 pre-mRNA (Vuillaumier-Barrot et al. Hum Mutat. 1999;14(6);543-544; Gonazlez-Dominguez et al., Mol Genet and Metab Rep 2021, 28; 100781).
[0743] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, and/or 8, skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, and/or 8, of PMM2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide. [0744] In some embodiments, the antisense oligonucleotide comprises the sequence 5'- TAGCTGCAAAGCAAGTGAAGCGGAC -3' (SEQ ID NO: 150) or 5’- ATCACAAACACAACCTACCTCAGGC-3’ (SEQ ID NO: 151) to target the PMM2 gene (Table 2).
FD
[0745] A congenital neurodevelopmental disease is familial dysautonomia (OMIM 223900) which is characterized by unusually low numbers of neurons in the sensory and autonomic nervous systems. The resulting symptoms of patients include gastrointestinal dysfunction, scoliosis, and pain insensitivity. This disease is especially prevalent in the Ashkenazi Jewish population, where 1/3600 live births present familial dysautonomia. The genetic cause of familial dysautonomia was localized to a dysfunctional region spanning 177 kb on chromosome 9q31. The IKBKAP gene, one of the five genes identified in that region, was found to have a single-base mutation in over 99.5% of cases of observed familial dysautonomia (Slaugenhaupt SA et al. American Journal of Human Genetics. 68 (3): 598-605; U.S. Patent No. 10,344,282, herein incorporated by reference in its entirety). The single-base mutation within the IKBKAP gene, is a transition from cytosine to thymine, and is present in the 5’ splice donor site of intron 20 in the IKBKAP pre-mRNA. This prevents recruitment of splicing machinery, and thus exon 19 is spliced directly to exon 21 in the final mRNA product - exon 20 is removed from the pre-mRNA with the introns.
[0746] In some embodiments, the methods and compositions described herein are useful for treating familial dysautonomia, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in IKBKAP pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 target regions of the IKBKAP gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 20 target region of the IKBKAP pre-mRNA designated as an annealing site. [0747] In some embodiments, antisense oligonucleotides of the disclosure target IKBKAP pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and/or 37, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and/or 37, the disrupted reading frame is restored to an in-frame mutation. While familial dysautonomia is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 19, 20, or 26 of IKBKAP pre-mRNA (Axelrod and Gold-von Simson, Orph J of Rare Dis, 2007, 2:39; Dietrich and Dragatsis, Genet Mol Biol 2016, 39(4):497-514).
[0748] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and/or 37, skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, and/or 37, of IKBKAP pre- mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0749] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs: 152-156 to target the IKBKAP gene (Table 2).
PBD1A
[0750] Zellweger syndrome (PBD1A; OMIM 214100) is caused by homozygous or compound heterozygous mutation in the PEX1 gene on chromosome 7q21-q22. Zellweger syndrome is an autosomal recessive systemic disorder characterized clinically by severe neurologic dysfunction, craniofacial abnormalities, and liver dysfunction, and biochemically by the absence of peroxisomes. There are two common PEX1 gene mutations found in people with Zellweger spectrum disorder. One mutation replaces the amino acid glycine with the amino acid aspartic acid at position 843 in Pexlp (written as Gly843Asp or G843D). This mutation leads to reduced levels of the protein. Individuals who have the G843D mutation tend to have signs and symptoms that are at the less-severe end of the condition spectrum. The other common mutation, which is known as the 1700fs mutation, leads to the production of an abnormally short, nonfunctional Pexlp. Individuals who have the 1700fs mutation often have signs and symptoms that are at the severe end of the condition spectrum. Mutations in the PEX1 gene that cause Zellweger spectrum disorder reduce or eliminate the activity of the Pexlp protein. Without enough functional Pexlp, enzymes are not properly imported into peroxisomes. As a result, cells contain empty peroxisomes that cannot carry out their usual functions. The severe end of the condition spectrum is caused by the absence of functional peroxisomes within cells. The less severe end of the condition spectrum results from mutations that allow some peroxisomes to form (European Pat. Appl. No. EP4104867 A2, incorporated herein by reference in its entirety).
[0751] In some embodiments, the methods and compositions described herein are useful for treating Zellweger syndrome, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in PEX1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, target regions of the PEX1 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 10 target region of the PEX1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 13 target region of the PEX1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 14 target region of the PEX1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 18 target region of the PEX1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 19 target region of the PEX1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 20 target region of the PEX1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 21 target region of the PEX1 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2). [0752] In some embodiments, antisense oligonucleotides of the disclosure target PEX1 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, the disrupted reading frame is restored to an in-frame mutation. While Zellweger syndrome is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 10, 13, 14, 18, 19, 20, or 21 of PEX1 pre-mRNA (Crane et al., 2005, Hum Mutat 26(3): 167-175).
[0753] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, and/or 23, skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, ofPEXl pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0754] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:328-330 to target the PEXlgene (Table 2).
MRD23
[0755] Autosomal dominant mental retardation-23 (MRD23; 615761) is caused by heterozygous mutation in the SETD5 gene on chromosome 3p25. Mental retardation, autosomal dominant 23 (MRD23) is a disorder characterized by significantly below average general intellectual functioning associated with impairments in adaptive behavior and manifested during the developmental period. MRD23 patients manifest moderate to severe intellectual disability with additional variable features of brachycephaly, a low hairline, depressed nasal bridge, prominent high nasal root, tubular nose, upslanting palpebral fissures, long and smooth philtrum, micrognathia, thin upper lip, and crowded teeth. Behavioral problems, including obsessive- compulsive disorder, hand flapping with ritualized behavior, and autism, are prominent features. The disease is caused by mutations affecting the gene represented in this entry (European Pat. Appl. No. EP4104867 A2). [0756] In some embodiments, the methods and compositions described herein are useful for treating MRD23, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in SETD5 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, target regions of the SETD5 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 4 target region of the SETD5 pre- mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 4 target region of the SETD5 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 5 target region of the SETD5 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0757] In some embodiments, antisense oligonucleotides of the disclosure target SETD5 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, the disrupted reading frame is restored to an in-frame mutation. While MRD23 is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 4, 5, 7, 9, or 14 of SETD5 pre-mRNA (European Pat. Appl. No. EP4104867 A2; Crippa et al., 2020, Front Neurol 11 :631; Kuechler et al., 2014, Eur J of Genet 23:753-760).
[0758] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, of SETD5 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide. [0759] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- gccuccacag auucaggg-3’ (SEQ ID NOs:324) to target the SETD5 gene (Table 2).
EPM5
[0760] Epilepsy, progressive myoclonic 5 (EPM5; 607459) is a neurodegenerative disorder characterized by myoclonic seizures and variable neurologic symptoms including cognitive decline and persistent movement abnormalities. In some cases, a heterozygosity for a complex mutation in the PRICKLE2 gene, a 443G-A transition, resulting in an Argl48-to-His (R148H) substitution, and a 457G-A transition, resulting in a Vall53-to-Ile (V153I) substitution can be identified in a progressive myoclonic epilepsy patient. In some cases, a heterozygous 1813G-T transversion in the PRICKLE2 gene, resulting in a Val605-to-Phe (V605F) substitution can be identified in a progressive myoclonic epilepsy patient (European Pat. Appl. No. EP4104867 A2).
[0761] In some embodiments, the methods and compositions described herein are useful for treating EPM5, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in PRICKLE2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, or 8 target regions of the PRICKLE2 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 4 target region of the PRICKLE2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 5 target region of the PRICKLE2 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0762] In some embodiments, antisense oligonucleotides of the disclosure target PRICKLE2 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, and/or 8, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, and/or 8, the disrupted reading frame is restored to an in-frame mutation. While EPM5 is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 4, or 5 pre-mRNA (European Pat. Appl. No. EP4104867 A2). [0763] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, and/or 8 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, and/or 8 of PRICKLE2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0764] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- agggaguugc aauaucga-3’ (SEQ ID NOs:323) to target the PRICKLE2 gene (Table 2).
FHM1
[0765] At least 20 mutations in the CACNA1A gene have been identified in people with familial hemiplegic migraine type 1 (FHM1; OMIM 141500). FHM1 is characterized by an aura of hemiplegia that is always associated with at least one other aura symptom (e g., hemianopsia, hemisensory deficit, aphasia). Most of the mutations that cause FHM1 change single amino acids in the CaV2.1 channel. The most common mutation, which has been found in more than a dozen affected families, replaces the amino acid threonine with the amino acid methionine at protein position 666 (European Pat. Appl. No. EP4104867 A2).
[0766] In some embodiments, the methods and compositions described herein are useful for treating FHM1, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in CACNA1A pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, or 47 target regions of the CACNA1A gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 14 target region of the CACNA1A pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 15 target region of the CACNA1A pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 29 target region of the CACNA1A pre- mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 30 target region of the CACNA1A pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 36 target region of the CACNA1A pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 37 target region of the CACNA1A pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0767] In some embodiments, antisense oligonucleotides of the disclosure target CACNA1A pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and/or 47 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and/or 47, the disrupted reading frame is restored to an in-frame mutation. While FHM1 is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 14, 15, 29, 30, 36, or 37 pre-mRNA (European Pat. Appl. No. EP4104867 A2).
[0768] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and/or 47 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, and/or 47 of CACNA1A pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0769] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NO:s:341-346 to target the CACNA1A gene (Table 2). AHC
[0770] Alternating hemiplegia of childhood (OMIM 104290) is an autosomal dominant condition. Alternating hemiplegia of childhood can result from new mutations in the gene and occur in people with no history of the disorder in their family. The primary feature of this condition is recurrent episodes of temporary paralysis, often affecting one side of the body (hemiplegia). The known ATP1A2 gene mutation associated with this condition replaces a single amino acid in Na+/K+ ATPase: the amino acid threonine is replaced with the amino acid asparagine at protein position 378. This genetic change can impair the protein’s ability to transport ions (European Pat. Appl. No. EP4104867 A2).
[0771] In some embodiments, the methods and compositions described herein are useful for treating alternating hemiplegia of childhood, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in ATP1 A2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 target regions of the ATP1A2 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 22 target region of the ATP1A2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 23 target region of the ATP1A2 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0772] In some embodiments, antisense oligonucleotides of the disclosure target ATP1 A2 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23, the disrupted reading frame is restored to an in-frame mutation. While alternating hemiplegia of childhood is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 22, or 23 pre-mRNA (European Pat. Appl. No. EP4104867 A2). [0773] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and/or 23 of ATP1A2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0774] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- ggcgcagaac caccaggu-3’ (SEQ ID NOs:322) to target the ATP1 A2 gene (Table 2).
AGS 6
[0775] Aicardi-Goutieres syndrome-6 (AGS6; OMIM 615010) can be caused by homozygous, compound heterozygous, or heterozygous mutation in the ADAR gene on chromosome lq21.3. Aicardi-Goutieres syndrome (AGS) manifests as an early-onset encephalopathy that usually, but not always, results in severe intellectual and physical handicap. A subgroup of infants with AGS present at birth with abnormal neurologic findings, hepatosplenomegaly, elevated liver enzymes, and thrombocytopenia, a picture highly suggestive of congenital infection. Otherwise, most affected infants present at variable times after the first few weeks of life, frequently after a period of apparently normal development. Typically, they demonstrate the subacute onset of a severe encephalopathy characterized by extreme irritability, intermittent sterile pyrexias, loss of skills, and slowing of head growth. Over time, as many as 40% develop chilblain skin lesions on the fingers, toes, and ears. It is becoming apparent that atypical, sometimes milder, cases of AGS exist, and thus the true extent of the phenotype associated with mutation of the AGS-related genes is not yet known. For example, mutation of ADAR has recently been associated with a clinical presentation of acute bilateral striatal necrosis (European Pat. Appl. No. EP4104867 A2).
[0776] In some embodiments, the methods and compositions described herein are useful for treating AGS6, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in ADAR pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 target regions of the ADAR gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 2 target region of the ADAR pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 3 target region of the ADAR pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0777] In some embodiments, antisense oligonucleotides of the disclosure target ADAR pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and/or 15 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and/or 15, the disrupted reading frame is restored to an in-frame mutation. While AGS6 is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 2, or 3 pre-mRNA (European Pat. Appl. No. EP4104867 A2).
[0778] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and/or 15 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and/or 15 of ADAR pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodi ami date morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0779] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:317-321 to target the ADAR gene (Table 2).
EIEEF4
[0780] Epileptic encephalopathy, early infantile, 4 (EIEE4; OMIM 612164) is a severe form of epilepsy characterized by frequent tonic seizures or spasms beginning in infancy with a specific EEG finding of suppression-burst patterns, characterized by high-voltage bursts alternating with almost flat suppression phases. Affected individuals can have neonatal or infantile onset of seizures, profound mental retardation, and MRI evidence of brain hypomyelination. In some cases, in a patient with early infantile epileptic encephalopathy-4, a heterozygous 1631G-A transition in the STXBP1 gene, resulting in a gly544-to-Asp (G544D) substitution can be identified. (European Pat. Appl. No. EP4104867 A2).
[0781] In some embodiments, the methods and compositions described herein are useful for treating EIEEF4, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in STXBP1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 target regions of the STXBP1 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 6 target region of the STXBP1 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 7 target region of the STXBP1 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0782] In some embodiments, antisense oligonucleotides of the disclosure target STXBP1 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and/or 19 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and/or 19, the disrupted reading frame is restored to an in-frame mutation. While EIEEF4 is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 6, or 7 pre- mRNA (European Pat. Appl. No. EP4104867 A2).
[0783] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and/or 19 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and/or 19 of STXBP1 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide. [0784] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- GCCAGUGCCC AUAGCGGG-3’, or 5’-CUUAUGCCAG UGCCCAUA-3’ (SEQ ID NOs:331- 332) to target the STXBP1 gene (Table 2).
VWM
[0785] Leukoencephalopathy with vanishing white matter (VWM) (OMIM 603896) can be caused by homozygous or compound heterozygous mutation in any of the 5 genes encoding subunits of the translation initiation factor EIF2B: EIF2B1 on chromosome 12q24, EIF2B2 on chromosome 14q24, EIF2B3 on chromosome lp34, EIF2B4 on chromosome 2p23, or EIF2B5 on chromosome 3q27. VMW is an autosomal recessive neurologic disorder characterized by variable neurologic features, including progressive cerebellar ataxia, spasticity, and cognitive impairment associated with white matter lesions on brain imaging. The neurologic signs include progressive cerebellar ataxia, spasticity, inconstant optic atrophy, and relatively preserved mental abilities. Disease is chronic-progressive with, in most individuals, additional episodes of rapid deterioration following febrile infections or minor head trauma. The mode of inheritance is autosomal recessive (European Pat. Appl. No. EP4104867 A2).
[0786] In some embodiments, the methods and compositions described herein are useful for treating VWM, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in EIF2B5, EIF2B2, or EIF2B1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 target regions of the EIF2B5 gene; to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, or 9 target regions of the EIF2B2 gene, or; to at least one of exon 1, 2, 3, 4, 5, 6, 7, or 8 target regions of the EIF2B1 gene, and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, or 9, or; at least one of exon 1, 2, 3, 4, 5, 6, 7, or 8, respectively. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 12 target region of the EIF2B5 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 13 target region of the EIF2B5 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 6 target region of the EIF2B2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 1 target region of the EIF2B1 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0787] In some embodiments, antisense oligonucleotides of the disclosure target EIF2B5 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16; target EIF2B2 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, or 9; or disclosure target EIF2B1 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, or 8, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16, exon 1, 2, 3, 4, 5, 6, 7, 8, or 9, or exon 1, 2, 3, 4, 5, 6, 7, or 8, the disrupted reading frame is restored to an in-frame mutation. While Leukodystrophy with VWM is comprised of several genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 12 or 13 of the EIF2B5 pre-mRNA, exon 6 of the EIF2B2 pre- mRNA, or exon 1 of the EIF2B1 pre-mRNA (European Pat. Appl. No. EP4104867 A2).
[0788] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 of EIF2B5 pre-mRNA. In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 11, 2, 3, 4, 5, 6, 7, 8, or 9 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, or 9 of EIF2B2 pre- mRNA. In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, or 8 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, or 8 of EIF2B1 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0789] In some embodiments, the antisense oligonucleotide is SEQ ID NOs:325 to target the EIF2B5 gene. In some embodiments, the antisense oligonucleotide sequence is SEQ ID NOs:326 to target the EIF2B2 gene. In some embodiments, the antisense oligonucleotide sequence is SEQ ID NOs:327 to target the EIF2B1 gene (Table 2). ICCA, BFIE, andEKDl
[0790] Infantile Convulsions and paroxysmal Choreoath etosis (ICCA; OMIM 602066) syndrome is a neurological condition characterized by the occurrence of seizures during the first year of life and choreoathetotic dyskinetic attacks during childhood or adolescence. Mutations in the PRRT2 gene, located on 16pl 1.2, has recently been found in families affected by ICCA syndrome. Benign familial infantile epilepsy (BFIE; OMIM 607745) is a genetic epileptic syndrome characterized by the occurrence of afebrile repeated seizures in healthy infants, between the third and eighth month of life. BFIE is a genetically heterogeneous disease. In the majority of cases, mutations in the proline-rich transmembrane protein 2 (PRRT2) gene located at 16p 11.2 has been found. Episodic kinesigenic dyskinesia 1 (EKD1; OMIM 128200) can be referred to as familial paroxysmal kinesigenic dyskinesia. EKD1 is a disorder characterized by episodes of abnormal movement that range from mild to severe. In some cases, a heterozygous 1-bp duplication (649dupC) in exon 2 of the PRRT2 gene in the proline-rich domain, resulting in a frameshift and introduction of a stop codon 7 amino acids downstream of the insertion (Arg217ProfsTer8) can be identified. In some cases, a heterozygous 4-bp deletion (514delTCTG) in exon 2 of the PRRT2 gene in the proline-rich domain, resulting in a frameshift and premature termination can be identified. In some cases, a heterozygous 1-bp deletion (972delA) in exon 3 of the PRRT2 gene, resulting in a frameshift and premature termination in the second transmembrane motif can be identified. (European Pat. Appl. No. EP4104867 A2).
[0791] In some embodiments, the methods and compositions described herein are useful for treating ICCA, BFIE, or EKD1, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in PRRT2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, or 3 target regions of the PRRT2 gene and induce exon skipping in at least one of exon 1, 2, or 3. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 6 target region of the PRRT2 pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 1 target region of the PRRT2 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2). [0792] In some embodiments, antisense oligonucleotides of the disclosure target PRRT2 pre-mRNA and induces skipping of exon 1, 2, and/or 3 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, and/or 3 , the disrupted reading frame is restored to an in-frame mutation. While ICCA, BFIE, or EKD1, are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 1 or 2 pre-mRNA (European Pat. Appl. No. EP4104867 A2).
[0793] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, and/or 3 skipping is designed to be complementary to a specific target sequence within exon 1, 2, and/or 3 of PRRT2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0794] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:333-335 to target the PRRT2 gene (Table 2).
ALGS
[0795] Alagille syndrome (ALGS; OMIM 118450), also known as arteriohepatic dysplasia, is a rare, debilitating, autosomal dominant, multisystem disorder (Turnpenny and Ellard, Eur. J. Hum. Gen. 2012, 20, 251-257). Patients suffer from liver damage caused by abnormalities in the bile ducts. Other effects include heart disease, vascular anomalies, skeletal anomalies, ophthalmic features, facial features, renal anomalies, growth retardation, and pancreatic insufficiency. The reported ALGS prevalence of 1:70,000 is thought to be an underestimate because of the variability and reduced penetrance of the condition. Mutations of genes involved in Notch signaling have been reported to cause ALGS. Mutations in JAG1 cause ALGS type 1, while mutations in NOTCH2 cause ALGS type 2, which is less prevalent than ALGS type 1. JAG1 encodes JAG1 protein, a cell surface ligand for the Notch transmembrane receptors. Binding of JAG1 protein to the Notch receptors triggers a signaling cascade that results in transcription of genes involved in cell fate determination and differentiation (U.S. Pat. No. 11,096,956, herein incorporated by reference in its entirety). [0796] In some embodiments, the methods and compositions described herein are useful for treating ALGS, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in JAG1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 target regions of the JAG1 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 13 target region of the JAG1 pre-mRNA designated as an annealing site (U.S. Pat. No. 11,096,956).
[0797] In some embodiments, antisense oligonucleotides of the disclosure target JAG1 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and/or 26 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and/or 26, the disrupted reading frame is restored to an in-frame mutation. While ALGS are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 13 pre-mRNA (U.S. Pat. No. 11,096,956).
[0798] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and/or 26 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and/or 26 of JAG1 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0799] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:285-294 to target the JAG1 gene (Table 2).
TSC
[0800] Tuberous sclerosis complex (TSC; OMIM 191092) is a disorder characterized by growth of benign tumors in multiple organ systems (Au, K., et al., J. Child Neurol., 2004, 19: 699- 709). Tumors of the central nervous system (CNS) are the leading io cause of morbidity and mortality, followed by renal disease. Patients can suffer from abnormalities of the brain that may include seizures, intellectual disability, and developmental delay, as well as abnormalities of the skin, lung, kidneys, and heart. The disorder affects as many as 25,000 to 40,000 15 individuals in the United States and about 1 to 2 million individuals worldwide, with an estimated prevalence of one in 6,000 newborns. TSC is a genetic disorder with an autosomal dominant inheritance pattern, caused by inherited defects or de novo 20 mutations that occur on two genes, TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present. The TSC1 gene, on chromosome 9, produces a protein called hamartin. The TSC2 gene, discovered in 1993, is on chromosome 16 and produces the protein tuberin. Scientists 25 believe these proteins act in a complex as growth suppressors by inhibiting the activation of a master, evolutionarily conserved kinase called mTOR. Loss of regulation of mTOR occurs in cells lacking either hamartin or tuberin, and this leads to abnormal differentiation and development, and to 30 the generation of enlarged cells, as are seen in TSC brain lesions (U.S. Pat. No. 11,096,956).
[0801] In some embodiments, the methods and compositions described herein are useful for treating TSC, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in TSC2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 target regions of the TSC2 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 4 target region of the TSC2 pre-mRNA designated as an annealing site (U.S. Pat. No. 11,096,956).
[0802] In some embodiments, antisense oligonucleotides of the disclosure target TSC2 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and/or 40 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and/or 40, the disrupted reading frame is restored to an in-frame mutation. While TSC are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 4 pre-mRNA (U.S. Pat. No. 11,096,956).
[0803] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and/or 40 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and/or 40 of TSC2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0804] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:295-304 to target the TSC2 gene (Table 2).
USH
[0805] Usher syndrome (USH, or just 'Usher1; OMIM 276901) and nonsyndromic retinitis pigmentosa (NSRP) are degenerative diseases of the retina. The hearing impairment in Usher patients is mostly stable and congenital and can be partly compensated by hearing aids or cochlear implants. The degeneration of photoreceptor cells in Usher and NSRP is progressive and often leads to complete blindness between the third and fourth decade of life, thereby leaving time for therapeutic intervention. Mutations in the USH2A gene are the most frequent cause of Usher syndrome type Ila explaining up to 50% of all Usher patients worldwide (±1300 patients in the Netherlands) and, as indicated by McGee et al. (2010. J Med Genet 47(7):499-506), also the most prevalent cause of NSRP in the USA, likely accounting for 12-25% of all cases of retinitis pigmentosa (RP). The mutations are spread throughout the seventy-two USH2A exons and their flanking intron sequences, and consist of nonsense and missense mutations, deletions, duplications, large rearrangements, and splicing variants. Exon 13 is by far the most frequently mutated exon with two founder mutations (c.2299deIG (p.E767SfsX21) in USH2 patients and c.2276G>T (p.C759F) in NSRP patients). For exon 50, fifteen pathogenic mutations have been reported, of which at least eight are clearly protein-truncating. Also, a deep intronic mutation in intron 40 of USH2A (c.7595-2144A>G) was reported (Vache et al. 2012. Human Mutation 33(1)404-108), which creates a cryptic high-quality splice donor site in intron 40 resulting in the inclusion of an aberrant exon of 152 bp (Pseudo Exon 40, or PE40) in the mutant USH2A mRNA, that causes premature termination of translation (U.S. Pat. Appl. No. 2022021348 Al, herein incorporated by reference in its entirety).
[0806] In some embodiments, the methods and compositions described herein are useful for treating USH, by delivering an antisense oligonucleotide capable of inducing skipping of an exon in USH2A pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 target regions of the USH2A gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 13 target region of the USH2A pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 50 target region of the USH2A pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 62 target region of the USH2A pre-mRNA designated as an annealing site (U.S. Pat. Appl. No. 2022021348 Al).
[0807] In some embodiments, antisense oligonucleotides of the disclosure target USH2A pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, and/or 72 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and/or 72, the disrupted reading frame is restored to an in-frame mutation. While USH are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 13, 50, or 62 pre-mRNA (U.S. Pat. Appl. No. 2022021348 Al).
[0808] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and/or 72 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, and/or 72 of USH2A pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0809] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:305-316 to target the USH2A gene (Table 2).
PTHS
[0810] Pitt-Hopkins syndrome (PTHS; OMIM 610954) is characterized by mental retardation, wide mouth and distinctive facial features, and intermittent hyperventilation followed by apnea. PTHS is linked to haploinsufficiency of the TCF4 transcription factor gene. At least 50 mutations in the TCF4 gene have been found to cause Pitt-Hopkins syndrome. Some mutations delete a nucleotide within the TCF4 gene, while other mutations delete the TCF4 gene as well as a number of genes that surround it. Still other TCF4 gene mutations replace single nucleotides. The size of the mutation does not appear to affect the severity of the condition; individuals with large deletions and those with single nucleotide changes seem to have similar signs and symptoms. TCF4 gene mutations disrupt the protein’s ability to bind to DNA and control the activity of certain genes. These gene mutations typically do not affect the TCF4 protein’s ability to bind to other proteins. The TCF4 protein’s inability to bind to DNA and control the activity of certain genes, particularly those genes involved in nervous system development and function, contributes to the signs and symptoms of PTHS. It is also likely that the loss of the normal proteins that are attached to the nonfunctional TCF4 proteins contribute to the features of this condition (European Pat. Appl.
No. EP4104867 A2).
[0811] In some embodiments, the methods and compositions described herein are useful for treating PTHS, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in TCF4 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 target regions of the TCF4 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 10 target region of the TCF4 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0812] In some embodiments, antisense oligonucleotides of the disclosure target TCF4 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20, the disrupted reading frame is restored to an in-frame mutation. While Pitt-Hopkins Syndrome are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 10 pre-mRNA (European Pat. Appl. No. EP4104867 A2).
[0813] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 20 of TCF4 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0814] In some embodiments, the antisense oligonucleotide comprises the sequence selected from SEQ ID NOs:338-340 to target the TCF4 gene (Table 2). SMS
[0815] Smith-Magenis syndrome (SMS; OMIM 182290) is caused in most cases (90%) by a 3.7-Mb interstitial deletion in chromosome 17pl l.2. The disorder can also be caused by mutations in the RAI1 gene, which is within the Smith-Magenis chromosome region. Smith- Magenis syndrome is a developmental disorder that affects many parts of the body. The major features of this condition include mild to moderate intellectual disability, delayed speech and language skills, distinctive facial features, sleep disturbances, and behavioral problems. Affected individuals may have eye abnormalities that cause nearsightedness (myopia) and other vision problems. Although less common, heart and kidney defects also have been reported in people with Smith-Magenis syndrome. A small percentage of individuals with Smith-Magenis syndrome have a mutation in the RAI1 gene instead of a chromosomal deletion. Although these individuals have many of the major features of the condition, they are less likely than people with a chromosomal deletion to have short stature, hearing loss, and heart or kidney abnormalities (European Pat. Appl. No. EP4104867 A2).
[0816] In some embodiments, the methods and compositions described herein are useful for treating Smith-Magenis Syndrome, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in RAI1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, or 6 target regions of the RAI1 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, or 6. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 4 target region of the RAI1 pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0817] In some embodiments, antisense oligonucleotides of the disclosure target RAI1 pre- mRNA and induces skipping of exon 1, 2, 3, 4, 5, and/or 6 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, and/or 6, the disrupted reading frame is restored to an in-frame mutation. While Smith-Magenis Syndrome are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 4 pre-mRNA (European Pat. Appl. No. EP4104867 A2). [0818] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, and/or 6 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, and/or 6 of RAI1 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0819] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- UUCUUGGCAG CUGGAACA-3’ (SEQ ID NO:337) to target the RAI1 gene (Table 2).
GEFSP9
[0820] Generalized epilepsy with febrile seizures plus-9 (OMIM 616172) is an autosomal dominant neurologic disorder characterized by onset of febrile and/or afebrile seizures in early childhood, usually before age 3 years. Seizure types are variable and include generalized tonic- clonic, atonic, myoclonic, complex partial, and absence. Most patients have remission of seizures later in childhood with no residual neurologic deficits, but rare patients may show mild developmental delay or mild intellectual disabilities. In some cases, in a patient with GEFSP9 a heterozygous C.166C-T transition in the STX1B gene, resulting in a gln56-to-ter (Q56X) substitution can be identified (European Pat. Appl. No. EP4104867 A2).
[0821] In some embodiments, the methods and compositions described herein are useful for treating GEFSP9, by delivering an antisense oligonucleotide capable of inducing skipping of an exon in STX1B pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target regions of the STX1B gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 6 target region of the STX1B pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 7 target region of the STX1B pre-mRNA designated as an annealing site (European Pat. Appl. No. EP4104867 A2).
[0822] In some embodiments, antisense oligonucleotides of the disclosure target STX1B pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1 , 2, 3, 4, 5, 6, 7, 8, 9, and/or 10, the disrupted reading frame is restored to an in-frame mutation. While Generalized epilepsy with febrile seizures plus-9 are comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 6 or 7 pre-mRNA (European Pat. Appl. No. EP4104867 A2).
[0823] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 of STX1B pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0824] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- CUUCCGGGAC AGUGUGGA-3’ (SEQ ID NO:336) to target the STX1B gene (Table 2).
HGPS
[0825] Children with Hutchinson-Gilford progeria syndrome (HGPS; OMIM 176670) suffer from dramatic acceleration of some symptoms associated with normal aging, most notably cardiovascular disease that eventually leads to death from myocardial infarction andor stroke usually in their second decade of life. For the vast majority of cases, a de novo point mutation in the lamin A (LMNA) gene is the cause of HGPS. This missense mutation creates a cryptic splice donor site that produces a mutant lamin A protein, termed “progerin,” which carries a 50-aa deletion near its C terminus (Varga et al., 2006, PNAS 103(9):3250-3255; U.S. Pat. No. 8258109 B2, herein incorporated by reference in its entirety).
[0826] In some embodiments, the methods and compositions described herein are useful for treating HGPS, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in LMNA pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 target regions of the LMNA gene and induce exon skipping in at least one of exon 11, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 6 target region of the LMNA pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 7 target region of the LMNA pre-mRNA designated as an annealing site (U.S. Pat. No. 8258109 B2).
[0827] In some embodiments, antisense oligonucleotides of the disclosure target LMNA pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12, the disrupted reading frame is restored to an in-frame mutation. While HGPS is comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 6 or 7 pre-mRNA (U.S. Pat. No. 8258109 B2).
[0828] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12 of LMNA pre- mRNA. In some embodiments, the antisense oligomer is a phosphorodi ami date morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0829] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:276-284) to target the LMNA gene (Table 2).
FTDP-17
[0830] The MAPT (Microtubule associated protein tau) gene consists of 16 exons and its expression is regulated by complex alternative splicing. This results in the production of two types of alternatively spliced transcripts: one bearing Exon 10, also known as 4R (Four microtubule repeats) isoform and the other that lacks Exon 10 is called 3R isoform (Three microtubule repeats). Equal levels of these two isoforms are expressed in normal human adult brain. Though several mutations causing Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP- 17; OMIM 600274) are known in MAPT, a half of these affect alternative splicing of Exon 10. These include mis-sense mutations, silent mutations and point mutations which are located in Exon 10, introns 9 and 10. They are known to implicate an increase in Exon 10 causing an excessive accumulation of 4R. This leads to the formation of neurofibrillary tangles, hence resulting in neurodegeneration (U.S. Pat. Appl. No. 20180066254 Al, herein incorporated by reference in its entirety).
[0831] In some embodiments, the methods and compositions described herein are useful for treating FTDP-17, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in MAPT pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 target regions of the MAPT gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 10 target region of the MAPT pre-mRNA designated as an annealing site (U.S. Pat. Appl. No. 20180066254 Al).
[0832] In some embodiments, antisense oligonucleotides of the disclosure target MAPT pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and/or 13 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and/or 13, the disrupted reading frame is restored to an in-frame mutation. While FTDP-17 is comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 10 pre-mRNA (U.S. Pat. Appl. No. 20180066254 Al).
[0833] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and/or 13 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and/or 13 of MAPT pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0834] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:386-388) to target the MAPT gene (Table 2).
PHMDS, andSCZD15
[0835] Phelan-McDermid Syndrome (PHMDS; OMIM 606232) is a developmental disorder with variable features. Common features include neonatal hypotonia, global developmental delay, normal to accelerated growth, absent to severely delayed speech, autistic behavior, and minor dysmorphic features. Other less common features associated with this syndrome included increased tolerance to pain, dysplastic toenails, chewing behavior, fleshy hands, dysplastic ears, pointed chin, dolichocephaly, ptosis, tendency to overheat, and epicanthic folds. Researchers have showed that Phelan-McDermid syndrome neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission (European Pat. Appl. No. 4104867 A2).
[0836] Susceptibility to schizophrenia 15 (SCZD15; OMIM 613950) has been associated with mutation in the SH3 and multiple ankyrin repeat domains-3 gene (SHANK3). Schizophrenia- 15 is a complex, multifactorial psychotic disorder or group of disorders characterized by disturbances in the form and content of thought (e.g. delusions, hallucinations), in mood (e.g. inappropriate affect), in sense of self and relationship to the external world (e.g. loss of ego boundaries, withdrawal), and in behavior (e.g bizarre or apparently purposeless behavior). Although it affects emotions, it is distinguished from mood disorders in which such disturbances are primary. Similarly, there may be mild impairment of cognitive function, and it is distinguished from the dementias in which disturbed cognitive function is considered primary. Some patients manifest schizophrenic as well as bipolar disorder symptoms and are often given the diagnosis of schizoaffective disorder (European Pat. Appl. No. 4104867 A2).
[0837] In some embodiments, the methods and compositions described herein are useful for treating PHMDS AND/OR SCZD15, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in SHANK3 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 target regions of the SHANK3 gene and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 10 target region of the SHANK3 pre-mRNA designated as an annealing site (U.S. Pat. Appl. No. 20180066254 Al). [0838] In some embodiments, antisense oligonucleotides of the disclosure target SHANK3 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22, the disrupted reading frame is restored to an in-frame mutation. While PHMDS and/or SCZD15 is comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 10 pre-mRNA (European Pat. Appl. No. 4104867 A2).
[0839] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22 of SHANK3 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0840] In some embodiments, the antisense oligonucleotide sequence is 5’- ACCACGUUCACCCCGUUC-3’ (SEQ ID NO:358) to target the SHANK3 gene (Table 2).
NF2
[0841] Neurofibromatosis type II (NF2; OMIM 101000) is caused by mutation in the gene encoding neurofibromin-2, which is also called merlin, on chromosome 22ql2.2. Neurofibromatosis type II is an inheritable disorder with an autosomal dominant mode of transmission. Incidence of the disease is about 1 in 60,000. Through statistics, it is suspected that one-half of cases are inherited, and one-half are the result of new, de novo mutations (European Pat. Appl. No. 4104867 A2).
[0842] In some embodiments, the methods and compositions described herein are useful for treating NF2, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in NF2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 target regions of the NF2 gene and induce exon skipping in at least one of exon 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 10 target region of the NF2 pre-mRNA designated as an annealing site (U.S. Pat. Appl. No. 20180066254 Al).
[0843] In some embodiments, antisense oligonucleotides of the disclosure target NF2 pre- mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16, the disrupted reading frame is restored to an in-frame mutation. While NF2 is comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 10 pre-mRNA (European Pat. Appl. No. 4104867 A2).
[0844] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 of NF2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0845] In some embodiments, the antisense oligonucleotide sequence is selected from SEQ ID NOs:351-357 to target the NF2 gene (Table 2).
PARKS
[0846] Parkinson’s Disease 8 (PARK8; OMIM 607060) is a progressive neurological disorder estimated to affect 7-10 million people worldwide. There is no treatment available that cures or slows the progression of PD. Elevated leucine-rich repeat kinase 2 (LRRK2) activity has been associated with genetic and sporadic forms of PD and, thus, reducing LRRK2 function is a promising therapeutic strategy (Korecka et al., Mol Therap Nucleic Acid 21 :623-635).
[0847] In some embodiments, the methods and compositions described herein are useful for treating PARK8, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in LRRK2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 target regions of the LRRK2 gene and in-duce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to exon 31, or 41 target region of the LRRK2 pre-mRNA designated as an annealing site (U.S. Pat. No 9,840,710, incorporated by reference herein in its entirety).
[0848] In some embodiments, antisense oligonucleotides of the disclosure target LRRK2 pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and/or 51 so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and/or 51, the disrupted reading frame is restored to an in- frame mutation. While PARK8 is comprised of few genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 31, or 41 LRRK2 pre-mRNA (U.S. Pat. No 9,840,710, incorporated by reference herein in its entirety).
[0849] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and/or 51skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and/or 51 of LRRK2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0850] In some embodiments, the antisense oligonucleotide sequence is selected from SEQ ID NOs:389-391 to target the LRRK2 gene (Table 2). 2. Musculoskeletal disorders
[0851] Multiple forms of muscular dystrophy are now known to be caused by defects in the O-linked glycosylation of a-dystroglycan. These dystroglycanopathies span a spectrum of phenotypes from Walker- Warburg syndrome (WWS; OMIM 236670; characterized by severe congenital muscular dystrophy, retinal and anterior chamber eye abnormalities, cobblestone lissencephaly, and hydrocephalus) to mild, adult onset LGMD. Included in this group of disorders is Fukuyama congenital muscular dystrophy (FCMD; OMIM 253800). Patients with typical FCMD display dystrophic changes in skeletal muscle, structural brain malformations, and severe ocular abnormalities. Most patients are never able to walk independently and have moderate to severe cognitive delay. The average life span is less than 20 years (Puckett et al., Further evidence ofFukutin mutations as a cause of childhood onset limb-girdle muscular dystrophy without mental retardation, Neurosmucul Disord. 2009 May;19(5):352-356).
DMD
[0852] Dystrophin-associated muscular dystrophies range from the severe Duchenne muscular dystrophy (DMD; OMIM 310200) to the milder Becker muscular dystrophy (BMD; OMIM 300376). Mapping and molecular genetic studies showed that both are the result of mutations in the huge gene that encodes dystrophin, also symbolized as DMD. Approximately two-thirds of the mutations in both forms are deletions of one or many exons in the dystrophin gene. Although there is no clear correlation found between the extent of the deletion and the severity of the disorder, DMD deletions usually result in frameshift.
[0853] In some embodiments, the methods and compositions described herein are useful for treating Duchenne muscular dystrophy (DMD), by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in myostatin (MSTN) pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 1, 2, or 3 target regions of the MSTN gene and induce exon skipping in at least one of exon 1, 2, or 3. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 2 target region of the MSTN pre-mRNA designated as an annealing site (PCT Pub. No. WO2022212886). [0854] Tn some embodiments, antisense oligonucleotides of the disclosure target MSTN pre-mRNA and induces skipping of exon 1, 2, and/or 3, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, and/or 3, the disrupted reading frame is restored to an in-frame mutation. While DMD is comprised of various genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 1, 2, and/or 3 of MSTN pre-mRNA. In some embodiments, DMD mutations amenable to skipping exon 2 comprise a subgroup of DMD patients.
[0855] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, and/or 3 skipping is designed to be complementary to a specific target sequence within exon 1 , 2, and/or 3 of MSTN pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0856] In some embodiments, the antisense oligonucleotide comprises the sequence 5'- AGCCCATCTTCTCCTGGTCCTGGGAAGG-3' (SEQ ID NO: 157) to target the MSTN gene (Table 2).
[0857] In some embodiments, the methods and compositions described herein are useful for treating Duchenne muscular dystrophy (DMD), by delivering an antisense oligonucleotide capable of inducing skipping of an exon in DMD pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to at least one of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 target regions of the dystrophin gene and induce exon skipping in at least one of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, and 63. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 23 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 43 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 43 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 44 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 45 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 50 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 51 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 52 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 53 target region of the dystrophin pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 55 target region of the dystrophin pre-mRNA designated as an annealing site (U.S. Pat. No. 11,193,125 B2; U.S. Pat. Appl. No. 20210147839A1; U.S. Pat. Appl. No. 20210222169A1; U.S. Pat. Appl. No. 20220152086 Al; U.S. Pat. Appl. No. 20220333112A1; U.S. Pat. Appl. No. 20210008095 Al; U.S. Pat. Appl. No. 20200362336 Al; PCT Pub. No. WO2021172498 Al; U.S. Pat. Appl. No. 20230038956 Al; all of which are incorporated by reference herein in their entirety).
[0858] In some embodiments, antisense oligonucleotides of the disclosure target dystrophin pre-mRNA and induces skipping of exon 23, 43, 44, 45, 50, 51, 52, 53, and / or 55, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 23, 43, 44, 45, 50, 51, 52, 53, and / or 55, the disrupted reading frame is restored to an in-frame mutation. While DMD is comprised of various genetic subtypes, antisense oligonucleotides of the disclosure were specifically designed to skip exon 23, 43, 44, 45, 50, 51, 52, 53, and / or 55 of dystrophin pre-mRNA. In some embodiments, DMD mutations amenable to skipping exon 43 comprise a subgroup of DMD patients (<5%). In some embodiments, DMD mutations amenable to skipping exon 44 comprise a subgroup of DMD patients (<5%). In some embodiments, DMD mutations amenable to skipping exon 45 comprise a subgroup of DMD patients (8%). In some embodiments, DMD mutations amenable to skipping exon 50 comprise a subgroup of DMD patients (<5%). In some embodiments, DMD mutations amenable to skipping exon 51 comprise a subgroup of DMD patients (13%). In some embodiments, DMD mutations amenable to skipping exon 52 comprise a subgroup of DMD patients (<5%). In some embodiments, DMD mutations amenable to skipping exon 53 comprise a subgroup of DMD patients (10%). In some embodiments, DMD mutations amenable to skipping exon 53 comprise a subgroup of DMD patients (<5%).
[0859] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 23, 43, 44, 45, 50, 51, 52, 53, and / or 55 skipping is designed to be complementary to a specific target sequence within exon 23, 43, 44, 45, 50, 51, 52, 53, and / or 55 of dystrophin pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0860] In some embodiments, the antisense oligonucleotide comprises the sequence 5'- GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO: 99). In some embodiments where the antisense oligonucleotide is a peptide nucleic acid (PNA) oligonucleotide, the antisense oligonucleotide comprises the sequence 5'-KKKGGCCAAACCTCGGCTTACCTGAAATKKK- 3' (SEQ ID NO:405), where K is lysine.
[0861] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs: 157-222, SEQ ID NOs:395-405, or SEQ ID NOs:410-988 to target the DMD gene (Table 2 and Table 3).
[0862] In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 9,228,187, entitled “Antisense molecules and methods for treating pathologies,” which is hereby incorporated by reference. In some embodiments, the antisense nucleotide composition includes any sequence disclosed in U.S. Patent Number 9,447,415, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 9,758,783, entitled “Antisense molecules and methods for treating pathologies,” which is hereby incorporated by reference. In some embodiments, the antisense nucleotide composition includes any sequence disclosed in U.S. Patent Number 10,287,586, entitled “Antisense molecules and methods for treating pathologies,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,781,450, entitled “Antisense molecules and methods for treating pathologies”, which is hereby incorporated by reference.
[0863] In some embodiments, the antisense oligonucleotide is selected from the group consisting of: (i) an antisense oligonucleotide of 34 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (ii) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (iii) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre- mRNA, wherein the target region is annealing site H45A (-06+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (iv) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-12+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (v) an antisense oligonucleotide of 22 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (vi) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (vii) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-12+16), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (viii) an antisense oligonucleotide of 32 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-14+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (ix) an antisense oligonucleotide of 27 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-08+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (x) an antisense oligonucleotide of 32 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-07+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xi) an antisense oligonucleotide of 34 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-12+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xii) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xiii) an antisense oligonucleotide of 39 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+30), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xiv) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-06+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xv) an antisense oligonucleotide of 34 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-06+28), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xvi) an antisense oligonucleotide of 25 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; and (xvii) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+28), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; or a pharmaceutically acceptable salt thereof.
[0864] In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 22 bases comprising the base sequence CAA UGC CAU CCU GGA GUU CCU G (SEQ ID NO:395), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide and is uniformly modified to comprise a 5-substituted pyrimidine base, or a pharmaceutically acceptable salt thereof. In other embodiments, the antisense oligonucleotide is the preceding antisense oligonucleotide, wherein the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. In other embodiments, the antisense oligonucleotide is chemically linked to a polyethylene glycol chain. In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 34 bases comprising the base sequence GCC CAA UGC CAU CCU GGA GUU CCU GUA AGA UAC C (SEQ ID NO:396), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide and is uniformly modified to comprise a 5-substituted pyrimidine base, or a pharmaceutically acceptable salt thereof. The antisense oligonucleotide of claim 35, wherein the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. In other embodiments, the antisense oligonucleotide is chemically linked to a polyethylene glycol chain. In further embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 31 bases comprising the base sequence GCC CAA UGC CAU CCU GGA GUU CCU GUA AGA U (SEQ ID NO:397), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide and is uniformly modified to comprise a 5-substituted pyrimidine base, or a pharmaceutically acceptable salt thereof. In other embodiments, the antisense oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the antisense oligonucleotide. In other embodiments, the antisense oligonucleotide is chemically linked to a polyethylene glycol chain. In other embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 39 bases comprising the base sequence UUG CCG CUG CCC AAU GCC AUC CUG GAG UUC CUG UAA GAU (SEQ ID NO:398), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide and is uniformly modified to comprise a 5-substituted pyrimidine base, or a pharmaceutically acceptable salt thereof (U.S Pat. No. 9,228,187).
[0865] In some embodiment, the antisense oligonucleotide of 20 to 31 bases comprises a base sequence that is 100% complementary to consecutive bases of exon 45 of the human dystrophin pre-mRNA, wherein the base sequence comprises at least 20 consecutive bases of CCA AUG CCA UCC UGG AGU UCC UGU AA (SEQ ID NO: 192), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide induces exon 45 skipping; or a pharmaceutically acceptable salt thereof.
[0866] In some embodiments, the antisense oligonucleotides are used in a method for treating a patient with Duchenne muscular dystrophy (DMD) in need thereof who has a mutation of the DMD gene that is amenable to exon 45 skipping, comprising administering to the patient an antisense oligonucleotide selected from the group consisting of (i) an antisense oligonucleotide of 34 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (ii) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (iii) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-06+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (iv) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-12+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (v) an antisense oligonucleotide of 22 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (vi) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre- mRNA, wherein the target region is annealing site H45A (-09+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (vii) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-12+16), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (viii) an antisense oligonucleotide of 39 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-14+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (ix) an antisense oligonucleotide of 27 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-08+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (x) an antisense oligonucleotide of 32 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-07+25), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xi) an antisense oligonucleotide of 34 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre- mRNA, wherein the target region is annealing site H45A (-12+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xii) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xiii) an antisense oligonucleotide of 39 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-09+30), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xiv) an antisense oligonucleotide of 28 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-06+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xv) an antisense oligonucleotide of 34 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-06+28), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; (xvi) an antisense oligonucleotide of 25 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+22), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; and (xvii) an antisense oligonucleotide of 31 bases in length 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A (-03+28), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping; or a pharmaceutically acceptable salt thereof, thereby treating the patient.
[0867] In some embodiments, the antisense oligonucleotide is of 22 bases in length, wherein the antisense oligonucleotide is 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A(-03+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide has the IUPAC chemical structure P-DEOXY-P-(DIMETHYLAMINO)) (2',3'-DIDEOXY-2',3'- IMINO-2',3'-SECO) (2'A-5')(C-A-A-M5U-G-C-C-A-M5U-C-C-M5U-G-G-A-G-M5U-M5U-C- C-M5U-G), 5'-(P-(4-((2-(2-(2-HYDROXYETHOXY)ETHOXY)ETHOXY)CARBONYL)-l- PIPERAZINYL)-N,N-DIMETHYLPHOSPHON AMID ATE (SEQ ID NO: 399)
[0868] In some embodiments, the antisense oligonucleotides are used in a method for restoring an mRNA reading frame to induce dystrophin protein production in a patient with Duchenne muscular dystrophy (DMD) in need thereof who has a mutation of the DMD gene that is amenable to exon 45 skipping, comprising administering to the patient an antisense oligonucleotide of 22 bases in length, wherein the antisense oligonucleotide is 100% complementary to a target region of exon 45 of the human dystrophin pre-mRNA, wherein the target region is annealing site H45A(-03+19), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the annealing site inducing exon 45 skipping, or a pharmaceutically acceptable salt thereof, thereby restoring the mRNA reading frame to induce dystrophin protein production in the patient.
[0869] In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 9,018,368, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 9,243,245, entitled “Means and methods for counteracting muscle disorders,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 9,506,058, entitled “Compositions for treating muscular dystrophy,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,337,003, entitled “Compositions for treating muscular dystrophy,” which is hereby incorporated by reference. In some embodiments, the antisense nucleotide composition includes any sequence disclosed in U.S. Patent Number 10,364,431, entitled “Compositions for treating muscular dystrophy”, which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,781,451, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof, ” which is hereby incorporated by reference.
[0870] In some embodiments, the antisense oligonucleotide of 30 bases comprises the base sequence CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG (SEQ ID NO:207), in which the uracil bases are thymine bases (CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG, SEQ ID NO:208), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain. In a further embodiment, the antisense oligonucleotide is included in a pharmaceutical composition comprising an antisense oligonucleotide of 30 bases comprising the base sequence CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG (SEQ ID NO:207), in which the uracil bases are thymine bases (CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG, SEQ ID NO:208), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain, and a pharmaceutically acceptable carrier. In other embodiments, the antisense oligonucleotide is in a composition comprising: a first compound that increases the level of a functional dystrophin protein produced in a muscle cell of a Duchenne Muscular Dystrophy (DMD) or Becker Muscular Dystrophy (BMD) individual, wherein said first compound is an antisense oligonucleotide that induces skipping of exon 51 of human dystrophin pre-mRNA of said individual; and a second compound comprising a steroid; wherein, upon administration to a DMD or BMD patient, the composition increases the ratio of said dystrophin to laminin-a2 in muscle tissue of said patient as compared to the ratio of said dystrophin to laminin-a2 in muscle tissue of a patient administered with said first compound and not said second compound; and wherein said antisense oligonucleotide is 100% complementary to a portion of exon 51 that is 13 to 50 nucleotides in length and wherein said oligonucleotide comprises a non naturally-occurring modification. In other embodiments, the antisense oligonucleotides are used in a method for treating a patient with Duchenne muscular dystrophy (DMD) in need thereof who has a mutation of the DMD gene that is amenable to exon 51 skipping, comprising intravenously administering to the patient eteplirsen at a dose of about 30 mg/kg weekly for more than 120 weeks, such that disease progression in the patient is delayed, thereby treating the patient. In some embodiments, the antisense oligonucleotide is used in a method of treating Duchenne muscular dystrophy (DMD) in a human subject who has a mutation of the DMD gene that is amenable to exon 51 skipping, comprising administering to the human subject a composition comprising eteplirsen and a phosphate-buffered saline at a dose of eteplirsen of about 30 mg/kg to about 50 mg/kg for a period of time sufficient to increase the number of dystrophin-positive fibers in a subject to at least 20% of normal. In some embodiments, the antisense oligonucleotide is used in a method for treating Duchenne muscular dystrophy (DMD) in a patient in need thereof who has a mutation of the DMD gene that is amenable to exon 51 skipping, comprising intravenously administering to the patient a composition comprising eteplirsen, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, wherein eteplirsen, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 30 mg/kg once a week for more than 120 weeks, such that disease progression in the patient is delayed, thereby treating the patient. In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 30 bases comprising the base sequence CUC CAA CAU CAA GGA AGA UGG CAU UUC UAG (SEQ ID NO:207), in which the uracil bases are thymine bases (CTC CAA CAT CAA GGA AGA TGG CAT TTC TAG, SEQ ID NO:208), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide.
[0871] In some embodiments, the antisense oligonucleotide used to treat DMD has the IUPAC chemical structure P-DEOXY-P-(DIMETHYLAMINO))(2',3'-DIDEOXY-2',3'-IMINO- 2',3'-SECO)(2'a^5')(C-m5U-C-C-A-A-C-A-m5U-C-A-A-G-G-A-A-G-A-m5U-G-G-C-A-m5U- m5U-m5U-C-m5U-A-G),5'-(P-(4-((2-(2-(2-HYDROXYETHOXY)-ETHOXY)-ETHOXY)- CARBONYL)-! -PIPERAZINYL)-N,N-DIMETHYLPHOSPHON AMID ATE) (SEQ ID NO 400)
[0872] In some embodiments, the antisense oligonucleotide composition includes any sequence dis-closed in U.S. Patent Number 9,024,007, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 9,994,851, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,227,590, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,266,827, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,421,966, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof’, which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,968,450, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof’, which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,995,337, entitled “Antisense oligonucleotides for inducing exon skipping and methods of use thereof’, which is hereby incorporated by reference.
[0873] In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 25 bases comprising abase sequence that is 100% complementary to 25 consecutive nucleotides of a target region of exon 53 of the human dystrophin pre-mRNA, wherein the target region is within annealing site H53A(+23+47) and annealing site H53A(+39+69), wherein the antisense oligonucleotide base sequence comprises at least 20 consecutive bases of CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G (SEQ ID NO:212), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain, and wherein the antisense oligonucleotide specifically hybridizes to the target region to induce exon 53 skipping. In a further embodiment, the antisense oligonucleotide is included in a pharmaceutical composition comprising an antisense oligonucleotide of 25 bases comprising a base sequence that is 100% complementary to 25 consecutive nucleotides of a target region of exon 53 of the human dystrophin pre-mRNA, wherein the target region is within annealing site H53A(+23+47) and annealing site H53A(+39+69), wherein the antisense oligonucleotide base sequence comprises at least 20 consecutive bases of CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G (SEQ ID NO: 212), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain, and wherein the antisense oligonucleotide specifically hybridizes to the target region to induce exon 53 skipping, and a pharmaceutically acceptable carrier. In other embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 20 to 31 bases comprising a base sequence that is 100% complementary to consecutive bases of a target region of exon 53 of the human dystrophin pre-mRNA, wherein the target region is within an-nealing site H53A(+23+47) and annealing site H53A(+39+69), wherein the base sequence com-prises at least 12 consecutive bases of CUG AAG GUG UUC UUG UAC UUC AUC C (SEQ ID NO: 211), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide induces exon 53 skipping; or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 20 to 31 bases comprising a base sequence that is 100% complementary to consecutive bases of a target region of exon 53 of the human dystrophin pre- mRNA, wherein the base sequence comprises at least 12 consecutive bases of CUG AAG GUG UUC UUG UAC UUC AUC C (SEQ ID NO: 211), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide induces exon 53 skipping; or a pharmaceutically acceptable salt thereof. In other embodiments, the antisense oligonucleotide is used in a method for treating a patient with Duchenne muscular dystrophy (DMD) in need thereof who has a mutation of the DMD gene that is amenable to exon 53 skipping, comprising administering to the patient an antisense oligonucleotide of 20 to 31 bases comprising a base sequence that is 100% complementary to consecutive bases of a target region of exon 53 of the human dystrophin pre-mRNA, wherein the base sequence comprises at least 12 consecutive bases of CUG AAG GUG UUC UUG UAC UUC AUC C (SEQ ID NO:211), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide induces exon 53 skipping; or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide is an antisense oligonucleotide of 25 bases comprising a base sequence that is 100% complementary to consecutive bases of a target region of exon 53 of the human dystrophin pre-mRNA, wherein the base sequence comprises at least 12 consecutive bases of CUG AAG GUG UUC UUG UAC UUC AUC C (SEQ ID NO:211), in which uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, wherein the antisense oligonucleotide is chemically linked to a polyethylene glycol chain, and wherein the antisense oligonucleotide induces exon 53 skipping; or a pharmaceutically acceptable salt thereof. In other embodiments, the antisense oligonucleotide is an antisense oligonucleotide comprising a base sequence 25 bases in length that is 100% complementary to 25 consecutive nucleotide bases of a target region of exon 53 of the human dystrophin pre-mRNA, wherein the antisense oligonucleotide base sequence comprises at least 20 consecutive bases of CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G (SEQ ID NO:212), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the target region and induces exon 53 skipping, or a pharmaceutically acceptable salt thereof. In some embodiments, the antisense oligonucleotide is used in a method of treating Duchenne muscular dystrophy, comprising administering an effective amount of an antisense oligonucleotide comprising a base sequence 25 bases in length that is 100% complementary to 25 consecutive bases of a target region of exon 53 of the human dystrophin pre-mRNA, said morpholino antisense oligonucleotide is chemically linked to a polyethylene glycol chain; wherein the antisense oligonucleotide base sequence comprises at least 20 consecutive bases of CAU UCA ACU GUU GCC UCC GGU UCU GAA GGU G (SEQ ID NO: 212), in which the uracil bases are thymine bases, wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide, and wherein the antisense oligonucleotide specifically hybridizes to the target region and induces exon 53 skipping.
[0874] In some embodiments, the antisense oligonucleotide has the sequence GUU GCC UCC GGU UCU GAA GGU GUU C (SEQ ID NO:401). In further embodiments, the antisense oligonucleotide has the IUPAC chemical structure P-DEOXY-P-(DIMETHYLAMINO))(2',3'- DIDEOXY-2',3'-IMINO-2',3'-SECO)(2'A->5')(G-m5U-m5U-G-C-C-m5U-C-C-G-G-m5U-M5U- C-m5U-G-A-A-G-G-m5U-G-m5U-m5U-C), 5’-(P-(4-((2-(2-(2-HYDROXYETHOXY)-
ETHOXY-)ETHOXY)-C ARB ONYL)- 1 -PIPERAZINYL)-N,N- DIMETHYLPHOSPHON AMID ATE (SEQ ID NO:403). [0875] In some embodiments, the antisense oligonucleotide composition includes any sequence dis-closed in U.S. Patent Number 9,079,934, entitled “Antisense nucleic acids,” which is hereby incorporated by reference. In some embodiments, the antisense oligonucleotide composition includes any sequence disclosed in U.S. Patent Number 10,870,676, entitled “Antisense nucleic acids,” which is hereby incorporated by reference.
[0876] In some embodiments, the antisense oligonucleotide is an antisense oligomer which causes skipping of the 53rd exon in the human dystrophin gene, consisting of the nucleotide sequence of 5'-CCTCCGGTTCTGAAGGTGTTC-3' (SEQ ID NO:213), wherein the antisense oligomer is an oligonucleotide having the sugar moiety and/or the phosphate-binding region of at least one nucleotide constituting the oligonucleotide modified, or a morpholino oligomer. In other embodiments, the antisense oligonucleotide comprises the sugar moiety of at least one nucleotide constituting the oligonucleotide is a ribose in which the 2'-OH group is replaced by any one selected from the group consisting of OR, R, R'OR, SH, SR, NH2, NHR, NR2, N3, CN, F, Cl, Br and I (wherein R is an alkyl or an aryl and R' is an alkylene). In other embodiments, the antisense oligomer comprises the phosphate-binding region of at least one nucleotide constituting the oligonucleotide is any one selected from the group consisting of a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, a phosphoramidate bond and a boranophosphate bond. In other embodiments, the antisense oligonucleotide is included in a pharmaceutical composition for the treatment of muscular dystrophy, comprising as an active, ingredient the antisense oligonucleotide as defined above, or a pharmaceutically acceptable salt or hydrate thereof. In other embodiments, the antisense oligonucleotide is used in a method of treating Duchenne muscular dystrophy (DMD) in a patient in need thereof comprising administering an antisense oligonucleotide, or a pharmaceutically acceptable salt or hydrate thereof, wherein the antisense oligonucleotide consists of a nucleotide sequence complementary to the sequence consisting of the 36th to the 56th nucleotides from the 5’ end of the 53rd exon in a human dystrophin pre-mRNA.
[0877] In some embodiments, the antisense oligonucleotide has the sequence CCU CCG GUU CUG AAG GUG UUC (SEQ ID NO:402). In other embodiments, the antisense oligonucleotide has the IUPAC chemical structure P-DEOXY-P-(DIMETHYLAMINO))(2',3'- DIDEOXY-2',3'-IMINO-2',3,-SECO)(2'A->5,)(C-C-m5U-C-C-G-G-m5U-m5U-C-m5U-G-A-A- G-G-m5U-G-m5U-m5U-C) (SEQ ID NO:404). DM1 and DM2
[0878] Myotonic dystrophy type 1 (DM1; OMIM 160900) and type 2 (DM2; OMIM 602668) are associated with long polyCUG and polyCCUG repeats in the 3'-UTR and intron 1 regions of the transcript dystrophia myotonica protein kinase (DMPK) and zinc finger protein 9 (ZNF9), respectively. While normal individuals have as many as 30 CTG repeats, DM1 patients carry a larger number of repeats ranging from 50 to thousands. The severity of the disease and the age of onset correlates with the number of repeats. Patients with adult onsets show milder symptoms and have less than 100 repeats, juvenile onset DM1 patients carry as many as 500 repeats and congenital cases usually have around a thousand CTG repeats. The expanded transcripts containing CUG repeats form a secondary structure, accumulate in the nucleus in the form of nuclear foci and sequester RNAbinding proteins (RNA-BP) (U.S. Pat. No. 10,106,796, herein incorporated by reference in its entirety).
[0879] In some embodiments, the methods and compositions described herein are useful for treating myotonic dystrophy type 1 (DM1) or 2 (DM2), by delivering an antisense oligonucleotide capable of inducing skipping of an exon in DMPK or ZNF9 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to the 3’-UTR polyGUC repeats target region of the DMPK gene; or to the intron 1 polyCCUG repeats target region of the ZNF9 gene, and induce exon skipping in at least one of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the DMPK gene; or in at least one of exon 1, 2, 3, 4, or 5 of the ZNF9 gene.
[0880] In some embodiments, antisense oligonucleotides of the disclosure target DMPK pre-mRNA and induces skipping of exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the DMPK gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, the disrupted reading frame is restored to an in-frame mutation. In some embodiments, antisense oligonucleotides of the disclosure target ZNF9 pre-mRNA and induces skipping of exon 1, 2, 3, 4, or 5 of the ZNF9 gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 1, 2, 3, 4, or 5, the disrupted reading frame is restored to an in-frame mutation.
[0881] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 skipping is designed to be complementary to a specific target sequence within exon 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 of DMPK pre-mRNA. In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 1, 2, 3, 4, or 5 skipping is designed to be complementary to a specific target sequence within exon 1, 2, 3, 4, or 5 of ZNF9 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0882] In some embodiments, the antisense oligonucleotide comprises one of the sequence SEQ ID NOs:223-226 to target the DMPK gene. In some embodiments, the antisense oligonucleotide comprises one of the sequence SEQ ID NOs:227-232 to target the ZNF9 gene (Table 2).
SMA
[0883] Spinal muscular atrophy (SMA; OMIM 253400) is a neuromuscular disease caused by mutations in telomeric SMN1, a gene encoding a ubiquitously expressed protein (survival of motor neuron SMN) involved in spliceosome biogenesis. [0004] The SMN gene product is intracellular and SMN deficiency results in selective toxicity to lower motor neurons, resulting in progressive neuron loss and muscle weakness. The severity of the disease is modified by the copy number of a centromeric duplication of the homologous gene (SMN2), which carries a splice site mutation that results in production of only small amounts of the full length SMN transcript. Patients who carry one to two copies of SMN2 present with the severe form of SMA, characterized by onset in the first few months of life and rapid progression to respiratory failure. Patients with three copies of SMN2 generally exhibit an attenuated form of the disease, typically presenting after six months of age. Though many never gain the ability to walk, they rarely progress to respiratory failure, and often live into adulthood. Patients with four SMN2 copies may not present until adulthood with gradual onset of muscle weakness.
[0884] Accordingly, in some aspects a method of treating SMA in a subject (e.g., a human subject) having SMA involves administering to the subject a recombinant nucleic acid that encodes SMN1 (also referred to as a recombinant SMN1 gene), and an ASO that increases full-length SMN2 mRNA in a subject (also referred to as an SMN2 ASO) (U.S. Patent Appl. No. 20210308281 Al, herein incorporated by reference in its entirety). In some embodiments, the methods and compositions described herein are useful for treating Spinal muscular atrophy type 3, by delivering an antisense oligonucleotide capable of inducing skipping of an exon in SMN2 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 7 target regions of the SMN2 gene, and induce exon skipping in at least one of exon 7 the SMN2 gene.
[0885] In some embodiments, antisense oligonucleotides of the disclosure target SMN2 pre-mRNA and induces skipping of exon 7 of the SMN2 gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 7, the disrupted reading frame is restored to an in-frame mutation.
[0886] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 7 skipping is designed to be complementary to a specific target sequence within exon 7 of SMN2 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0887] In some embodiments, the antisense oligonucleotide comprises one of the sequence 5’- tcactttcataatgctgg-3’ (SEQ ID NO:233) to target the SMN2 gene.
IBMPFD1
[0888] In some embodiments, the methods and compositions described herein are useful for treating Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia 1 (IBMPFD1; OMIM 167320), by delivering an antisense oligonucleotide capable of inducing skipping of an exon in VCP pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 5 target regions of the VCP gene, and induce exon skipping in at least one of exon 5 the VCP gene.
[0889] In some embodiments, antisense oligonucleotides of the disclosure target VPC pre- mRNA and induces skipping of exon 5 of the VCP gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 5, the disrupted reading frame is restored to an in-frame mutation.
[0890] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 5 skipping is designed to be complementary to a specific target sequence within exon 5 of VCP pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0891] In some embodiments, the antisense oligonucleotide comprises one of the sequence SEQ ID NO s: 265 -270 to target the VCP gene.
3. Cardiovascular diseases
FH
[0892] Familial hypercholesterolemia (FH; OMIM 144010) is a condition characterized by extremely high concentrations of low-density lipoprotein (LDL) cholesterol, most commonly due to genetic defects in the hepatic LDL receptor. Patients with the most severe form, homozygous FH, develop life-threatening cardiovascular disease (CVD) in early adulthood. Lowering LDL cholesterol is known to reduce mortality and morbidity, delaying the onset of CVD (Disterer et al., Mol Therap. 2013 21(3);602-609; PCT Publication No. WO2013057485 Al).
[0893] In some embodiments, the methods and compositions described herein are useful for treating FH, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in APOB pre-mRNA carrying a deleterious mutation, e g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 27 target regions of the APOB gene, and induce exon skipping in at least one of exon 27 the APOB gene.
[0894] In some embodiments, antisense oligonucleotides of the disclosure target APOB pre-mRNA and induces skipping of exon 27 of the APOB gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 27, the disrupted reading frame is restored to an in-frame mutation. [0895] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 27 skipping is designed to be complementary to a specific target sequence within exon 27 of APOB pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0896] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:368-370 to target the APOB gene (Table 2).
4. Metabolic diseases
ACADMD
[0897] Medium-chain Acyl-CoA dehydrogenase (MCAD) deficiency (ACADMD; OMIM 201450) is the most commonly recognized defect of the mitochondrial P-oxidation in humans. It is a potentially fatal autosomal recessive inherited defect, which may present in the first 2 years of life if patients experience periods of metabolic stress to the P-oxidation system (Vianey-Liaud et al. 1987; Roe and Coates 1989). The clinical picture is heterogeneous, ranging from severe episodes of hypoglycemia and coma to years of remaining without symptoms (Roe and Coates 1989). Occasionally, patients with ACADMD die suddenly and unexpectedly (Roe and Coates 1989). Approximately 80% of patients with ACADMD are homozygous for the G985 mutation and can thus be easily diagnosed by a simple PCR-based assay (Gregersen et al. 1991b; Yokota et al. 1991). The majority of the remaining patients are compound heterozygotes with G985 as one of the disease alleles (Andresen et al., Disease -causing mutations in exon 11 of the medium chain Acyl-CoA dehydrogenase gene, Am. J. Hum. Genet 54:975-988, 1994; Holm et al., 2022, Hum Mutat 43(2) :253 -265).
[0898] In some embodiments, the methods and compositions described herein are useful for treating ACADMD, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in MCAD pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 5 target regions of the MCAD gene, and induce exon skipping in at least one of exon 5 the MCAD gene. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 11 target regions of the MCAD gene, and induce exon skipping in at least one of exon 11 the MCAD gene.
[0899] In some embodiments, antisense oligonucleotides of the disclosure target MCAD pre-mRNA and induces skipping of exon 5 of the MCAD gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 5, the disrupted reading frame is restored to an in-frame mutation. In some embodiments, antisense oligonucleotides of the disclosure target MCAD pre-mRNA and induces skipping of exon 11 of the MCAD gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 11, the disrupted reading frame is restored to an in-frame mutation.
[0900] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 5 skipping is designed to be complementary to a specific target sequence within exon 5 of MCAD pre-mRNA. In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 11 skipping is designed to be complementary to a specific target sequence within exon 11 of MCAD pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
PKU
[0901] Deficiency of phenylalanine hydroxylase (PAH, EC 1.14.16.1) is causing phenylketonuria (PKU, OMIM 261600), an autosomal recessively inherited disease presenting with elevated blood phenylalanine (Phe) levels. The phenotypic severity of PKU is characterized by the type of mutation, and thus by residual PAH enzyme activity. The fully functional homotetrameric PAH catalyzes hydroxylation of Phe to tyrosine (Tyr) in the presence of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and molecular oxygen. According to the Locus Knowledgebase (PAHdb, www.pahdb.mcgill.ca), about 60% of mutations in the PAH gene are missense mutations, which may lead to a misfolding of the protein, disturbing the complex enzyme regulation and changes in kinetics, due to altered affinities for the Phe substrate and the BH4 cofactor (Heintz et al., Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry, Mol Genet and Metab 2012 105(4);559-565). [0902] In some embodiments, the methods and compositions described herein are useful for treating phenylketonuria, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in PAH pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 27 target regions of the PAH gene, and induce exon skipping in at least one of exon 11 the PAH gene.
[0903] In some embodiments, antisense oligonucleotides of the disclosure target PAH pre- mRNA and induces skipping of exon 11 of the PAH gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 11, the disrupted reading frame is restored to an in-frame mutation.
[0904] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 11 skipping is designed to be complementary to a specific target sequence within exon 11 of PAH pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0905] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- ATCCTCTTTGGTAACCTCACCTCAC-3’ (SEQ ID NO:371) to target the PAH gene (Table 2).
NPC1
[0906] Niemann-Pick Disease, type IC (NPC1; OMIM 257220) is an autosomal recessive lipid storage disorder characterized by progressive neurodegeneration. Approximately 95% of cases are caused by mutations in the NPC1 gene, referred to as type Cl; 5% are caused by mutations in the NPC2 gene, referred to as type C2. The clinical manifestations of types Cl and C2 are similar because the respective genes are both involved in egress of lipids, particularly cholesterol, from late endosomes or lysosomes. Patients usually develop difficulty coordinating movements (ataxia), an inability to move the eyes vertically (vertical supranuclear gaze palsy), poor muscle tone (dystonia), severe liver disease, and interstitial lung disease. Individuals with NPC1 have problems with speech and swallowing that worsen over time, eventually interfering 16 with feeding. Affected individuals often experience progressive decline in intellectual function and about one-third have seizures.
[0907] In some embodiments, the methods and compositions described herein are useful for treating NPC1, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in NPC1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 27 target regions of the NPC1 gene, and induce exon skipping in at least one of exon 15 the NPC1 gene.
[0908] In some embodiments, antisense oligonucleotides of the disclosure target NPC1 pre-mRNA and induces skipping of exon 15 of the NPC1 gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 15, the disrupted reading frame is restored to an in-frame mutation.
[0909] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 15 skipping is designed to be complementary to a specific target sequence within exon 15 of NPC1 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0910] In some embodiments, the antisense oligonucleotide comprises the sequence 5’- UGGCAUCACGGACAAUGC -3’ (SEQ ID NO:341) to target the NPC1 gene (Table 2).
5. Cancers
RAS-associated cancers
[0911] The three closely related human RAS genes, HRAS, KRAS, and NRAS, are all widely expressed and are important for regulation of numerous cellular processes through the RAS-MAP-kinase and PI3K/Akt pathways. They each exhibit oncogenic activity and more than 30% of all human tumors have mutations leading to constitutively active RAS proteins. Different RAS oncogenes are preferentially associated with different types of human cancers. Therefore, the RAS oncogenes are already targets for numerous different anti cancer treatments (U.S. Patent No. 10,266,828, incorporated herein by reference in its entirety).
[0912] In some embodiments, the methods and compositions described herein are useful for treating various RAS-associated cancers including, but not limited to, bladder cancer (OMIM 109800), breast cancer (OMIM 114480), gastric cancer (OMIM 613659), acute myeloid leukemia (OMIM 601626), lung cancer (OMIM 211980), pancreatic carcinoma (OMIM 260350), RAS- associated autoimmune leukoproliferative disorder type IV (OMIM 614470), colorectal cancer (OMIM 114500), and follicular thyroid carcinoma (OMIM 188470), by delivering an antisense oligonucleotide capable of inducing skipping of an exon in HRAS, KRAS, or NRAS pre-mRNA carrying a deleterious mutation, e g. that causes a frameshift mutation. In some embodiments, antisense nucleotides of the disclosure are complementary to at least one of exon 1, 2, 3, 4, 5, or 6 target regions of the HRAS gene; at least one of exon 1, 2, 3, 4, or 5 target regions of the KRAS gene; at least one of exon 1 , 2, 3, 4, 5, 6, or 7 target regions of the NRAS gene; and induce skipping in at least one of exon 1, 2, 3, 4, 5, or 6 of the HRAS gene; 1, 2, 3, 4, or 5 of the KRAS gene; and 1, 2, 3, 4, 5, 6, or 7 of the NRAS gene, respectively. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 2 target region of the HRAS pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 2 target region of the KRAS pre-mRNA designated as an annealing site. In some embodiments, the disclosure relates to antisense oligonucleotides complementary to an exon 2 target region of the NRAS pre-mRNA designated as an annealing site.
[0913] In some embodiments, antisense oligonucleotides of the disclosure target HRAS pre-mRNA and induces skipping of exon 2, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 2, the disrupted reading frame is restored to an in-frame mutation. In some embodiments, antisense oligonucleotides of the disclosure target KRAS pre- mRNA and induces skipping of exon 2, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 2, the disrupted reading frame is restored to an in-frame mutation. In some embodiments, antisense oligonucleotides of the disclosure target NRAS pre- mRNA and induces skipping of exon 2, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 2, the disrupted reading frame is restored to an in-frame mutation. [0914] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 2 skipping is designed to be complementary to a specific target sequence within exon 2 of HRAS, KRAS, or NRAS pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0915] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:234-244 to target the HRAS gene. In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:245-255 to target the KRAS gene. In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:256-264 to target the NRAS gene.
MLD
[0916] Metachromatic leukodystrophy (MLD; OMIM 250100) is a lysosomal storage disease caused by an arylsulfatase A (ARSA) deficiency and characterized by severe neurological symptoms resulting from demyelination within the central and peripheral nervous systems. This disease is characterized pathologically by myelin degeneration in both the central and peripheral nervous systems (CNS and PNS). Although enzyme replacement therapy using human ARSA has been tried, this approach does not effectively relieve the neurological symptoms. Gene therapy is one of the potentially effective strategies under consideration for use in the treatment of CNS disorders, and several gene therapy protocols for treating MLD have been proposed (Miyake et al., 2021, Sci Rep 11 :20513).
[0917] In some embodiments, the methods and compositions described herein are useful for treating MLD, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in ARSA pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 2 target regions of the ARSA gene, and induce exon skipping in at least one of exon 2 the ARSA gene. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 3 target regions of the ARSA gene, and induce exon skipping in at least one of exon 3 the ARSA gene. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 4 target regions of the ARSA gene, and induce exon skipping in at least one of exon 4 the ARSA gene (European Pat. Appl. EP4104867 A2).
[0918] In some embodiments, antisense oligonucleotides of the disclosure target ARSA pre-mRNA and induces skipping of exon 2, 3, and/or 4 of the ARSA gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 2, 3, and/or 4, the disrupted reading frame is restored to an in-frame mutation.
[0919] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 2, 3, and/or 4 skipping is designed to be complementary to a specific target sequence within exon 2, 3, and/or 4 of ARSA pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0920] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:359-363 to target the ARSA gene (Table 2).
6. Luns disorders
CF
[0921] Cystic fibrosis (CF; 219700) is a common, severe autosomal recessive disease caused by mutations in the CFTR gene. The CFTR gene encodes for a chloride channel responsible for chloride transport in epithelial cells. The major manifestations of CF are in the lungs, with more than 90% mortality related to the respiratory disease. The disease in the respiratory tract is linked to the insufficient CFTR function in the airway epithelium (PCT Publication No. WO2021199029 Al, incorporated herein by reference in its entirety).
[0922] In some embodiments, the methods and compositions described herein are useful for treating cystic fibrosis, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in CTFR pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 24 target regions of the CTFR gene, and induce exon skipping in at least one of exon 24 the CTFR gene. (PCT Publication No. WO2021199029 Al).
[0923] In some embodiments, antisense oligonucleotides of the disclosure target CTFR pre-mRNA and induces skipping of exon 24 of the CTFR gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 24, the disrupted reading frame is restored to an in-frame mutation.
[0924] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 24 skipping is designed to be complementary to a specific target sequence within exon 24 of CTFR pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0925] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:364-367 to target the CTFR gene (Table 2).
7. Other diseases
IBS
[0926] The term "inflammatory bowel disease" (IBS17; OMIM 612261) means an inflammatory disease in bowel that involves Thl7 cells. Crohn's disease and Ulcerative colitis represent exemplary diseases of the inflammatory bowel disease.
[0927] In some embodiments, the methods and compositions described herein are useful for treating IBS17, by delivering an antisense oligonucleotide capable of inducing skipping of an exon in IL23R pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 9 target regions of the IL23R gene, and induce exon skipping in at least one of exon 9 the IL23R gene. (U.S Pat. No. 9,868,776, incorporated by reference herein in its entirety).
[0928] In some embodiments, antisense oligonucleotides of the disclosure target IL23R pre-mRNA and induces skipping of exon 9 of the IL23R gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 9, the disrupted reading frame is restored to an in-frame mutation. [0929] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces ex-on 9 skipping is designed to be complementary to a specific target sequence within exon 9 of IL23R pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate mor-pholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase in-cluding, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0930] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:379-385 to target the IL23R gene (Table 2).
DEB
[0931] Epidermolysis bullosa is a group of inherited mechanobullous disorders characterized by fragility of the skin within the cutaneous basement membrane zone, with considerable clinical and genetic heterogeneity, inherited either in an autosomal dominant or autosomal recessive fashion. Traditionally, EB has been divided into three broad categories based on the level of tissue separation, determined by diagnostic electron microscopy and/or immunoepitope mapping: the simplex forms of EB (EBS) demonstrate tissue separation within the basal keratinocytes at the bottom layer of epidermis; the junctional forms of EB (JEB) display cleavage within the lamina lucida in the dermoepi dermal basement membrane; and in the dystrophic forms (DEB; OMIM 131750), tissue separation occurs below the lamina densa within the upper papillary dermis. DEB is caused by mutations in COL7A1, on chromosomal region 3p21, encoding type VII collagen. (U.S. Pat. No. 9,340,783, incorporated herein by reference in its entirety).
[0932] In some embodiments, the methods and compositions described herein are useful for treating DEB, by delivering a conjugate comprising a 3E10 antibody or an antigen binding fragment thereof conjugated to an antisense oligonucleotide capable of inducing skipping of an exon in COL7A1 pre-mRNA carrying a deleterious mutation, e.g., that causes a frameshift mutation. In certain embodiments, antisense oligonucleotides of the disclosure are complementary to exon 73, 74, or 80 target regions of the COL7A1 gene, and induce exon skipping in at least one of exon 73, 74, or 80 the COL7A1 gene.
[0933] In some embodiments, antisense oligonucleotides of the disclosure target COL7A1 pre-mRNA and induces skipping of exon 73, 74, and/or 80 of the COL7A1 gene, so it is excluded or skipped from the mature, spliced mRNA transcript. By skipping exon 73, 74, and/or 80, the disrupted reading frame is restored to an in-frame mutation.
[0934] In some embodiments, the nucleobase sequence of an antisense oligonucleotides that induces exon 73, 74, and/or 80 skipping is designed to be complementary to a specific target sequence within exon 73, 74, and/or 80 of COL7A1 pre-mRNA. In some embodiments, the antisense oligomer is a phosphorodiamidate morpholino oligomer (PMO) wherein each morpholino ring of the PMO is linked to a nucleobase including, for example, nucleobases found in DNA (adenine, cytosine, guanine, and thymine). In some embodiments, the antisense oligomer is a PNA oligonucleotide.
[0935] In some embodiments, the antisense oligonucleotide comprises a sequence selected from SEQ ID NOs:372-378 to target the COL7A1 gene (Table 2).
B. Dosages and Administration
[0936] Administration of the suitable compositions provided herein can be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, intrathecal, intratumoral, intraocular, intravitreal, intraarterial, topical, intradermal, etc., administration. The route of administration, of course, depends, inter alia, on the AOC contained in the pharmaceutical composition.
[0937] The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patient’s size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind and stage of infection or disease, general health and other drugs being administered concurrently.
C. Uses
[0938] Uses of an AOC described herein are also contemplated. In some aspects, the present disclosure provides a use of an AOC described herein, or a pharmaceutical composition comprising an AOC described herein, for the treatment of a genetic disease or disorder in a subject in need thereof In some aspects, the present disclosure provides a use of AOCs described herein, or the pharmaceutical composition comprising an AOC described herein, for inducing exon skipping in a pre-mRNA transcript to generate a mRNA transcript encoding a truncated protein. VI. Kits
[0939] In some aspects, the disclosure encompasses kits comprising one or more containers and comprising one or more doses of a complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof disclosed herein. In certain embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising, for example, a complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof disclosed herein, with or without one or more additional agents. For other embodiments, such a unit dosage is supplied in single-use prefdled syringe for injection. In still other embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on, or associated with, the container(s) indicates that the enclosed composition is used for diagnosis or treatment.
[0940] The present invention also provides kits for producing single-dose or multi-dose administration units of a complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof disclosed herein and, optionally, one or more other diagnostic or therapeutic agents. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition that is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits will generally contain a pharmaceutically acceptable formulation of a complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof and, optionally, one or more other therapeutic agents in the same or different suitable containers. The kits may also contain other pharmaceutically acceptable formulations, for combination therapy.
[0941] More specifically the kits may have a single container that contains the complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof), with or without additional components, or they may have distinct containers for each desired agent. Alternatively, the complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof and any optional therapeutic agent of the kit may be maintained separately within distinct containers prior to administration to a patient. The kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate- buffered saline (PBS), Ringer's solution and dextrose solution.
[0942] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.
[0943] As indicated briefly above the kits may also contain a means by which to administer the complex formed between a therapeutic mRNA molecule and a 3E10 antibody or antigen binding fragment thereof and any optional components to the patient, e.g., one or more needles or syringes, from which the formulation may be injected or introduced into the patient.
EXAMPLES
Example 1 - Preparation of a 3E10 antibody oligonucleotide conjugate for exon skipping [0944] Conjugates of a 3E10 antibody to antisense oligonucleotides (ASOs) that target exon 23 of murine Duchenne muscular dystrophy (DMD) for exon skipping were generated. The ASOs were phosphorodiamidate morpholino oligomers (PMOs), which are uncharged payloads that do not interact with the CDRs of 3E10 (V66), potentially facilitating conjugation, and they had the sequence 5’-GGCCAAACCTCGGCTTACCTGAAAT-3’ (SEQ ID NO: 1045) (Figure 15).
[0945] The ASOs were linked to the 3E10 antibody using mal eimide, SATA-SPP, and DBCO linkers, as depicted in Figure 8. The drug-antibody-ratios (DARs) of the conjugates were determined using UV/Vis spectroscopy, as shown below.
[0946] All V66-PMO conjugates were shown to complex exogenous nucleic acid by gel complexation experiments (not shown).
Example 2 — 3E10-phosphorodiamidate morpholino oligomer (PMO) conjugates demonstrate dose-dependent exon skipping in vitro
[0947] The ability of the V66-PMO conjugates of Example 1 to mediate exon skipping was evaluated in differentiated C2C12 myotubules, and the results are shown in Figure 9. Both stable and cleavable V66-PMO conjugates caused dose-dependent exon-skipping in differentiated C2C12 myotubules at concentrations of 2.5 pM, 1.25 pM, 0.625 pM (Figure 9). The exon skipping efficiency was improved in the 3E10 (V66) conjugate with a SPP cleavable linker. Furthermore, the 3E10 (V66) conjugate with a SPP cleavable linker showed faint double-exon- skipping that did not follow dosing trend.
Example 3 - Molecular Modeling of 3 El 0 and Engineered Variants Thereof
[0948] Molecular modeling of 3E10 (Pymol) revealed a putative Nucleic Acid Binding pocket (NAB1) (Figures 11A-11B; unterlined in the heavy and light chain sequences below). Mutation of aspartic acid at residue 31 of CDR1 to asparagine increased the cationic charge of this residue and enhanced nucleic acid binding and delivery in vivo (3E10-D3 IN); mutation to arginine (3E10-D31R) further expanded the cationic charge; and mutation to lysine (3E10-D3 IK) changed charge orientation (Figure 11 A).
[0949] WT HEAVY CHAIN scFv SEQUENCE
E VQLVESGGGL VKPGGSRKLS CAASGFTFSD YGMHWVRQAP EKGLEWVAYI SSGSSTIYYA DTVKGRFTIS RDNAKNTLFL QMTSLRSEDT AMYYCARRGL LLDYWGOGTT LT VS (SEQ ID NO: 1020) [0950] LIGHT CHAIN scFv SEQUENCE
D IVLTQSPASL AVSLGQRATI SCRASKSVST SSYSYMHWYQ QKPGQPPKLL IKYASYLESG VPARFSGSGS GTDFTLNIHP VEEEDAATYY CQHSREFPWT FGGGTKLEIK RADAAPGGGG SGGGGSGGGGS (SEQ ID NO: 1021)
[0951] Figure 11B is an illustration showing molecular modeling of 3E10-scFv (Pymol) with NAB1 amino acid residues illustrated with punctate dots.
Example 4 — Single dose exon skipping in Duchenne muscular dystrophy (DMD) using a 3E10-phosphorodiamidate morpholino oligomer (PMO) conjugate
[0952] A non-cleavable conjugate of a 3E10 antibody to a PMO ASO that targets exon 23 of murine DMD for exon skipping was generated using a commercial kit and having the structure shown in the top panel of Figure 8 (3E10 (V66)-PMO). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15).
[0953] The DAR was determined to be 2, and ability of the 3E10 (V66)-PMO conjugate to mediate exon skipping was observed in vitro.
[0954] An mdx DMD mouse ((MDX Tg) C57BL/10ScSn-Dmdmdx/J , Jackson Strain #:001801) received a single dose at 4 mg/kg (oligo dose) of the 3E10 (V66)-PMO conjugate, and tissue was harvested 7 days later. As shown in Figure 10, exon skipping was observed in all muscles tested, i.e., Tibialis anterior (TA), Gastrocnemius (G), Quadricep (Q), Deltoid (D), and Heart (H).
Example 5 - Study design for confirmatory single dose exon skipping by 3E10 (V66) in mdx mice
[0955] A single dose IV study was designed to confirm DMD functional skipping in mdx (MDX Tg) C57BL/ 10ScSn-Dmdmdx/J , Jackson Strain #: 001801) mice with the non-cleavable 3E10 (V66) PMO conjugate tested in Example 4 and two additional conjugates, a second non-cleavable 3E10 (V66) conjugate having the structure shown in the middle panel of Figure 8, and a disulfide cleavable 3E10 (V66) PMO conjugate having the structure shown in the bottom panel of Figure 8. The two non-cleavable AOCs were administered IV at 5.5 mg/kg, and the cleavable AOC was administered by IP at 4 mg/kg (Figure 12). Exon-skipping was observed in all muscle tissue types and heart tissue in mice treated with the 3E10 (V66)-PMO conjugates after 7 days post-treatment (N=5 mice; Figures 14A and 14B). [0956] As shown in Figure 14A, a higher level and consistency of exon skipping was observed in muscle tissues in mdx mice receiving the 3E10 (V66) conjugate with a non-cleavable linker relative to mdx mice receiving the 3E10 (V66) conjugate with a disulfide, cleavable linker, even though dose levels varied (5.5 mg/kg vs 4 mg/kg) due to dosing limitations. DMD exon skipping was also observed in heart tissue, particularly in those animals treated with 3E10 (V66) conjugate with a non-cleavable linker. Variable exon skipping was detected in animals receiving PMO oligo only as a control in this study.
Example 6 - ENT2 is highly expressed in skeletal muscle
[0957] ENT2 protein levels were investigated in various tissues by Western blot. As shown in Figure 22, high ENT2 expression was confirmed in muscle at the protein level in healthy human and mouse tissues. Furthermore, significant ENT2 protein expression was also detected in heart and diaphragm tissue.
Example 7 - Labeled 3E10 (V66) conjugate with a phosphorodiamidate morpholino oligomer (PMO) is internalized in C2C12 muscle myotubes and A427 tumor cells
[0958] Cellular internalization of 3E10 (V66)-PMO conjugates having a non-cleavable mal eimide linker was investigated by live cell imaging. The PMO ASOs had the sequences of SEQ ID NOs: 138-143 (Figure 15). Time course experiments were performed at approximately at 0, 2.5, 5, 7.5, 10, 12.5, 15, and 17.5 hours after administration of the 3E10 (V66)-PMO conjugates to C2C12 muscle myotubes and A427 tumor cells. As shown in Figure 23, a labeled 3E10 (V66) - PMO conjugate having an ASO sequence of SEQ ID NO: 1100 (Figure 15) was readily internalized and transited rapidly to the nuclei of selected cells, avoiding lysosomal trafficking, and demonstrating and confirming efficient nuclear delivery by 3E10 (V66) conjugate to enable exon skipping of dystrophin pre-RNA in the nucleus. Furthermore, the uptake of the 3E10 (V66) - PMO into muscle cells (C2C12 muscle myotubes) in vitro was as efficient as its uptake into tumor cells (A427 tumor cells).
Example 8 - Single dose study with 3E10 (V66) — PMO conjugate demonstrates restoration of DYS expression in muscle tissue of mdx mice
[0959] An assessment of single (9 and 18 mg/kg) IV doses of a 3E10 (V66) -PMO conjugate with non-cleavable linker having the structure shown in the middle panel of Figure 8 was investigated by measuring exon skipping by Q-PCR and dystrophin restoration by Western blot. The PMO ASO had the sequence of SEQ ID NO:1 100 (Figure 15). The tissues assessed for exon skipping include Tibialis anterior (TA), Gastrocnemius (G), Quadricep (Q), Deltoid (D), and Heart (H).
[0960] PMO alone (18 mg/kg), 3E10 (V66) - PMO conjugate (9 mg/kg), and 3E10 (V66)
- PMO conjugate (18 mg/kg) were administered to mdx mice ((MDX Tg) C57BL/10ScSn- Dmdmdx/ Jackson Strain #:001801) by IV as a single dose on day 0 and exon skipping quantified as %A23/total DYS by Q-PCR at days 7, 14, and 28. The 3E10 (V66)-DBCO-PMO (DMD) drug- antibody ratio (DAR) was 2.7. As shown in Figure 25, administration of a single dose of the 3E10 (V66) - PMO conjugate (concentrations of 9 mg/kg and 18 mg/kg) demonstrated dose- and time- dependent increases in exon skipping. A single dose of the noncleavable 3E10 (V66) - PMO conjugate at 18 mg/kg achieved 10-15% exon skipping at day 14, and the dose-dependent increase in exon skipping that was observed peaked prior to day 28. Lastly, the delivery of the 3E10 (V66)
- PMO conjugate was consistently higher and more pronounced that that of PMO alone.
[0961] Next, Dystrophin restoration was investigated by Western blot, and a representative image is shown for day 14 deltoid and heart samples in Figure 26 for the 3E10 (V66) - PMO conjugate at a concentration of 18 mg/kg. Dystrophin protein expression was quantified for both 9 and 18 mg/kg dose levels, and for PMO alone, for both day 14 and day 28 samples, and the results shown in Figure 27. Restored dystrophin protein was observed in all skeletal muscles, heart, and diaphragm in all mdx mice treated with single dose IV administration of the 3E10 (V66) - PMO conjugate at both 9 and 18 mg/kg at day 14 and day 28.
[0962] As shown in Figure 27, single dose of 3E10 (V66) - PMO conjugate significantly improved dystrophin restoration (on average, a 10-fold difference), compared to delivery of PMO alone in both a dose- and time-dependent manner. Notably, consistent with the previously described exon skipping data, no dystrophin restoration was measured in heart for treatment with PMO alone.
Example 9 - Tryptic peptide mapping to identify preferential lysine residues for 3E10- D31N monoclonal antibody (V66) conjugation
[0963] The previous results demonstrate that a lysine conjugated 3E10 (V66) PMO conjugate can efficiently internalize into muscle cells and deliver the PMO payload to the nucleus, resulting in detectable dystrophin pre-RNA exon skipping and protein restoration in both skeletal and cardiac muscles following single dose systemic IV administration. Since the variable regions and CDRs of the 3E10-D3 IN monoclonal antibody (V66) contain multiple lysine residues, it was investigated whether conjugation in this region could occur, which may have a negative impact on antibody immunoreactivity and function.
[0964] 3E10-D31N monoclonal antibody (V66) Lys-azide conjugation intermediates were digested and mapped by mass spectrometry to characterize modified lysines in the VH and VL chains of the antibody and as shown in Figures 28A and 28B. As shown in Figure 28B, most levels of lysine modification are modest, and many of the modified lysines are not implicated in antibody function. As shown in Figure 28C, the light chain K53 was the most frequently modified lysine (nearly 2/3 of instances) and is in, or adjacent to (depending on the schema used to define the CDRs), CDR2. These results suggest that lysine conjugation may have a negative impact on immunoreactivity of a 3E10 AOC if light chain CDR2 residue K53 is modified.
Example 10 - Improved cellular internalization of 3E10-D3IN monoclonal antibody (V66) oligonucleotide conjugates utilizing transglutaminase-mediated enzymatic conjugation
[0965] To evaluate alternative conjugation methods of PMO that do not employ lysine linkages to 3E10 (V66), site-specific transglutaminase conjugation within the Fc region of the antibody was attempted using the non-cleavable linker design, and DAR 2 was successfully achieved. Cellular internalization of 3E10-D31N monoclonal antibody (V66) PMO ASO conjugates was investigated in A427 tumor cells comparing lysine versus transglutaminase- mediated enzymatic conjugation methods, and internalization was detected by fluorescent microscopy using a labeled secondary anti-Fc detection antibody. Both conjugates contained ASO with the sequence of SEQ ID NO: 1100 (Figure 15). These results are exemplified in Figures 29A and 29B, which show fold change in nuclei median fluorescence intensity (MFI) of all tested antibody oligonucleotide conjugate lots, normalized to antibody alone control. As shown in Figures 29A and 29B, transglutaminase-mediated enzymatic conjugation had greater MFI than the 3E10-D31N monoclonal antibody (V66) oligonucleotide conjugated with lysine intermediate, consistent with greater cellular internalization, suggesting that lysine conjugation may be exerting some negative effect on cellular uptake, and that use of an alternative conjugation chemistry may enhance the functional activity of 3E10 (V66) conjugates. Example 11 - Modifications to 3E10-D31N monoclonal antibody (V66) oligonucleotide conjugates effect greater exon skipping in C2C12 Myotubules
[0966] Lysine versus transglutaminase-mediated enzymatic conjugation methods were evaluated for 3E10-D31N monoclonal antibody (V66) -oligonucleotide conjugates (AOCs) to determine the effect on exon skipping in C2C 12 myotubules. The linker-PMO design was the same in both conjugates, and is shown in the middle panel of Figure 8. As shown in Figure 30A, 3E10- D31N (V66)-PMOs having transglutaminase-mediated enzymatic conjugation demonstrated improved exon skipping over 3E10-D31N (V66)-PMOs having lysine conjugation. Furthermore, as shown in Figure 30B, varying the length of the non-cleavable PEG linkers (e.g., PEG4, PEG8, and PEG12) did not significantly impact exon skipping. Lastly, as shown in Figure 30C, 3E10- D31N (V66)-PMOs having the cleavable linkers, protease Cathepsin-B (CathB) and SPDMV (disulfide), showed enhanced exon-skipping compared to 3E10-D31N (V66)-PMOs having non- cleavable linkers with transglutaminase-mediated enzymatic conjugation. Furthermore, 3E10- D31N (V66)-PMOs having the hindered disulfide linker (SPDMV) conjugate (Tg-SPDMV) demonstrated the highest level of exon-skipping (~15%), representing greater than 10X improvement over lysine-PEG8 AOC (via lysine conjugation).
Example 12 - Linker stability of 3E10-D31N monoclonal antibody (V66) - oligonucleotide conjugates (AOCs) in mouse serum
[0967] Linker stability was evaluated for several 3E10-D3 IN monoclonal antibody (V66)- oligonucleotide conjugates (AOCs) with a PMO designed to improve the therapeutic efficacy targeting DMD in mouse serum. The AOCs tested were: (1) DMD 3E10-D31N monoclonal antibody (V66)-SPP-PMO using lysine conjugation;
[0968]
[0969] SPP linker
[0970] (2) DMD 3E10-D31N monoclonal antibody (V66)-Az-DBCO-PEG8-VC-PAB-
PMO;
[0975] DBCO-PEG8-VC-PAB linker
[0976] (3) 3E10-D31N monoclonal antibody (V66) Az-DBCO-PEG8-PMO using transglutaminase (TGase) mediated enzymatic conjugation;
[0984] DBCO-PEG4-SPDMV
[0985] The results in Figure 31 A indicate that a non-cleavable linker, a di-methyl-hindered disulfide cleavable linker, and protease cleavable linker had greater stability over a single methyl- hindered disulfide linker following prolonged incubation at 37 degrees in mouse serum. Figure 3 IB shows a data table indicating the panel of antibody-oligonucleotide conjugates (AOCs) tested and their oligonucleotide to antibody ratios (OAR or DAR) with the percent intact AOC at 120 hours incubation.
[0986] Furthermore, Figure 32, shows the in vitro performance comparison of a panel of antibody-oligonucleotide constructs. The in vitro results suggest that transglutaminase-SPDMV constructs with a disulfide cleavable linker outperformed all other tested AOC constructs regarding analyte profile, immunoreactivity with nucleic acids, serum stability, cell uptake, and exon skipping in vitro.
[0987] Further assays will be carried out to look at emergence of free payload (indicative of instability) and of OAR stability. Furthermore, plate based ELISAs and chromatographic methods (HIC) will be utilized to elucidate changes in OAR over time.
Example 13 - Single high dose systemic treatment of mdx mice with 3E10 (V66) PMO conjugate
[0988] A non-cleavable conjugate of a 3E10 antibody to a PMO ASO that targets exon 23 of murine DMD for exon skipping was generated having the structure shown in the middle panel of Figure 8 (“AOC” or “3E10 AOC”; V66-lysine conjugate (noncleavable linker (D AR 3.6)). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15).
[0989] A study was undertaken to evaluate exon skipping and PMO quantification following single dose escalation of the AOC in the DMD mdx mouse model ((MDX Tg) C57BL/10ScSn-Dmdmdx/J , Jackson Strain #:001801). The mice were administered the AOC by IV at doses of 2.5, 5, 10, 20, and 30 mg/kg (oligo concentration). Gastrocnemius, diaphragm, heart, tibialis anterior, deltoid and quadriceps tissues were collected on Days 7 and 14.
[0990] Exon skipping of DMD preRNA was determined and quantified by Q-PCR. As shown in Figure 33, a clear dose-dependent increase in exon skipping was observed following single dose IV administration of the noncleavable AOC. The TA and Deltoid exhibited the highest levels of exon skipping - at the highest dose (30 mg/kg) a level of skipping greater than 30% was observed.
[0991] Quantification of delivered PMO was determined by hybridization ELISA in muscle tissue samples from treated mice. As shown in Figure 34, high levels of tissue PMO delivery was observed in all muscle tissues at 7 days post-treatment, including the heart, in a dose - dependent manner. Appreciably high levels of PMO were quantified and did not saturate even at the highest doses. Further, as shown in Figures 35A and 35B, the high levels of tissue PMO delivery were maintained at least 14 days post-treatment in all muscle tissues. Example 14 - Single high dose systemic treatment of mdx mice with 3E10 (V66) PMO conjugate
[0992] A non-cleavable conjugate of a 3E10 antibody to a PMO ASO that targets exon 23 of murine DMD for exon skipping was generated having the structure shown in the middle panel of Figure 8 (“AOC” or “3E10 AOC”; V66-lysine conjugate (noncleavable linker (D AR 3.3)). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15).
[0993] A study was undertaken to evaluate high dose administration of the AOC in the DMD mdx mouse model ((MDX Tg) C57BL/10ScSn-Dmdmdx/J , Jackson Strain #:001801). The mice were administered the AOC by IV at doses of 30 and 42 mg/kg (oligo concentration). Gastrocnemius, diaphragm, heart, tibialis anterior, deltoid and quadriceps tissues were collected on Days 10, 21, and 28.
[0994] Exon skipping of DMD preRNA was determined and quantified by Q-PCR. Exon skipping was evaluated at day 10 and at day 21 post-single dose IV administration to assess accumulation of skipped DMD mRNA over time. As shown in Figure 36, at 30 mg/kg single IV dose administration, levels of exon 23 skipped mRNA continued to accumulate from day 10 to day 21 across all muscle tissues tested at nearly the same rate, achieving up to 40% skipped mRNA in tissues, including the heart. As shown in Figure 38, at day 10 post treatment, significantly greater DMD exon 23 skipping was determined for 3E10 AOC (up to 30-fold greater) compared to PMO alone in all muscle tissues tested, including and notably the heart. No exon skipping was noted in heart tissue in PMO alone samples.
[0995] Quantification of delivered PMO was determined by hybridization ELISA in muscle tissue samples from treated mice. As shown in Figure 37, the AOC delivered approximately 100-fold more PMO to muscle tissue than PMO administration alone 10 days post- treatment.
[0996] Dystrophin protein restoration was determined by western blot of test article tissue to a titrated standard curve of control tissue protein and mdx tissue protein extracts. As shown in Figure 39, the AOC led to dose-dependent increases in dystrophin protein restoration at day 28 post-single IV dose. Dystrophin restoration in TA increased 8-fold with increasing AOC dose, and in heart, a greater than 10-fold increase was observed. [0997] Distribution and localization of restored dystrophin was determined by immunofluorescence in treated muscle sections, with C57BL/10ScSn mice (Strain #000476) serving as WT control. As shown in Figures 40A-40C, a significant distribution (approximately 50% of WT control) of restored dystrophin was observed in the diaphragm in mdx mice 28 days following high single dose administration of the AOC. The restored dystrophin in the diaphragm colocalized in the sarcolemma with laminin staining. This pattern was also observed to varying degrees in all other muscle tissues tested, including the heart.
Example 15 - Head-to-head comparison of cleavable versus noncleavable linker AOCs and impact of DAR on potency of noncleavable linker AOCs
[0998] A study was undertaken to compare a cleavable peptide linker to a non-cleavable linker for AOC potency in mediating exon skipping in the mdx mouse. Further, a comparison study was performed to evaluate the impact of DAR 2 vs 3.3 in potency of AOCs having a non-cleavable linker in vivo.
[0999] The first non-cleavable AOC had the structure shown in the middle panel of Figure 8 (Lys-AOC = V66-lysine conjugate). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15), and the AOC had a DAR of 3.3.
[1000] The second non-cleavable AOC had the structure shown in the middle panel of Figure 8 (TG-AOC = V66-transglutaminase conjugate). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15), and the AOC had a DAR of 2.0.
[1001] The cleavable AOC had the structure shown in Figure 41 (“Cathepsin B cleavable AOC” or “V66-transglutaminase valine-citrulline linker conjugate”). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15), and the AOC had a DAR of 1.9.
[1002] DMD mdx mice ((MDX Tg) C57BL/10Sn-Dmdmdx/J , Jackson Strain 01801) were administered the AOCs by IV at a dose of 15 mg/kg (oligo concentration). Gastrocnemius, diaphragm, heart, tibialis anterior, deltoid and quadriceps tissues were collected on Day 14 for exon skipping analysis, as determined and quantified by Q-PCR.
[1003] As shown in Figure 42, the AOC having the protease (Cathepsin B) cleavable linker (V66-transglutaminase valine-citrulline linker conjugate) was functionally superior to the AOC having the non-cleavable linker (V66-transglutaminase conjugate). Given the unique biology of the 3E10 antibody, which avoids degradative lysosomes, this was a surprising outcome, suggesting that protease linker cleavage may occur in the cytosol or nucleus during 3E10 AOC trafficking.
[1004] As shown in Figure 43, the AOC having a greater DAR (3.3 vs. 2.0; V66-lysine conjugate vs. V66-transglutaminase conjugate) led to significantly greater exon skipping in all muscle tissues for 3E10 AOCs having noncleavable linker conjugates. This data demonstrates the importance of drug load per antibody on functional outcomes for PMO delivery in mdx mice using 3E10 AOCs. Notably, the lysine conjugation of PMO to 3E10 antibody outperformed the transglutaminase conjugation of PMO to 3E10 antibody having a lower DAR, despite potential concerns from analytical testing suggesting that the lysine conjugation method may compromise uptake.
Example 16 - Head-to-head comparison of high DAR cleavable disulfide linker to noncleavable linker following repeat dose administration
[1005] A study was undertaken to compare the functional activity of a disulfide cleavable linker (SPDMV) to a noncleavable (PEG8) linker at DAR 3 following repeat dosing of the AOC.
[1006] The noncleavable AOC had the structure shown in the middle panel of Figure 8 (noncleavable (PEG8) Lys - AOC = V66-lysine conjugate). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15), and the AOC had a DAR of 3.8.
[1007] The cleavable AOC had the structure shown in the third panel of Figure 52 (Disulfide cleavable (SPDMV) Lys - AOC = V66-lysine). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15), and the AOC had a DAR of 3.4.
[1008] DMD mdx mice ((MDX Tg) C57BL/10Sn-Dmdmdx/J , Jackson Strain #:001801) were administered the AOCs by IV at three doses of 30 mg/kg (oligo concentration). Gastrocnemius, diaphragm, heart, tibialis anterior, deltoid and quadriceps tissues were collected on Day 28 for exon skipping analysis, as determined and quantified by Q-PCR, protein restoration as evaluated by western blot, and immunofluorescent tissue distribution analysis. In situ hybridization was also performed to detect and visualize PMO distribution throughout muscle tissue to assess the penetrance of the 3E10 AOC into muscle tissues.
[1009] As shown in Figure 44, the disulfide cleavable AOC outperformed the noncleavable AOC for exon skipping following repeat dose administration in mdx mice. Q-PCR determined that 3E10 disulfide cleavable AOC resulted in 40-60% skipping in diaphragm and skeletal muscles, while only approximately 10% skipping was detected in the heart with both cleavable and non- cleavable AOCs. Given the frequency of dosing, maximal skipping, including in the heart, may occur at a later time point, beyond 28 days.
[1010] As shown in Figure 45, the 3E10 disulfide cleavable AOC resulted in 10-15% restored dystrophin protein in the muscle tissues tested, which was typically more than twice the level detected in the tissues treated with the noncleavable AOC. These data confirm the superiority of the disulfide cleavable linker design for 3E10 AOC design. Given the frequency of dosing, maximal dystrophin protein restoration may occur at a later time point, beyond 28 days.
[1011] Immunofluorescent detection of dystrophin was performed, and quantification of 3E10 AOC dystrophin detection was normalized to WT dystrophin and laminin colocalization to establish percent protein distribution in tissues. As shown in Figure 46, repeat dosing of the disulfide cleavable AOC resulted in 10-60% dystrophin protein distribution to sarcolemma in mdx mouse muscle tissues.
[1012] As shown in Figure 47, extensive and widespread PMO distribution was observed in the deltoid muscle of mice that were administered the noncleavable AOC, as determined by in situ hybridization (ISH). These results visibly confirm the robust high level of PMO delivery by 3E10 AOC to muscle tissue previously quantified by hybridization ELISA.
[1013] As shown in Figure 48, PMO delivered by the noncleavable AOC was broadly distributed throughout the muscle following systemic administration, with extensive nuclear delivery evident. These results show that a high dose of noncleavable 3E10 AOC deeply penetrated into deltoid muscle tissue, proximal to nearly all muscle fibers. The extensive nuclear staining observed is indicative of potent nuclear homing, and delivery of the payload by 3E10 AOC was evident, as highlighted by the arrows in Figure 48.
[1014] As shown in Figure 49, widespread tissue and cell nuclei delivery of PMO by 3E10 into heart muscle was observed for both the noncleavable AOC and the disulfide cleavable AOC. Evaluation of cardiac tissue confirmed that both noncleavable and disulfide cleavable treated 3E10 AOC mice showed strong nuclear staining for PMO in the heart tissue as determined by ISH. Example 17 - Head-to-head comparison of 3E10 AOC to PPMO and PMO alone following single dose administration
[1015] A study was carried out to compare PMO delivery and distribution in muscle tissues following a single IV dose at 10 mg/kg in DMD mdx mice ((MDX Tg) C57BL/10Sn-Dmdmdx/J , Jackson Strain #:001801).
[1016] The AOC had the structure shown in the third panel of Figure 52 (Disulfide cleavable (SPDMV) Lys - AOC = V66-lysine). The ASO had the sequence of SEQ ID NO: 1100 (Figure 15), and the AOC had a DAR of 3.4.
[1017] As shown in Figure 50, at Day 7 post-administration of 10 mg/kg single IV dose, the disulfide cleavable 3E10 AOC delivered more than lOx more PMO than PPMO alone, and lOOx more than naked PMO alone, including in heart tissue.
[1018] As shown in Figure 51, the disulfide cleavable 3E10 AOC delivered significantly more PMO to muscle tissue and muscle nuclei compared to PMO or PPMO alone. Very limited detection was observed for PMO and PPMO treated tissues. For the disulfide cleavable 3E10 AOC, the staining pattern was well dispersed and extensive across most muscle fibers, with intense staining in multiple nuclei, demonstrating the superior tissue and nuclear targeting from the disulfide cleavable 3E10 AOC.
Example 18- Linker optimization of 3E10 A OCs
[1019] Further investigation was performed to determine the optimal linker, conjugation site, and DAR for maximal 3E10 AOC functional output. Cleavable linkers had been determined to be superior to non-cleavable linker for covalent linkage of the DMD exon-skipping PMO, with the hindered disulfide (SPDMV) linker being the more optimal linker. Additional linkers, including phosphatase (PO4)- and glucuronidase-cleavable linkers were tested (Figure 52). While the phosphatase cleavable linker performed comparable to the disulfide cleavable linker, the glucuronidase cleavable linker performed poorly and was not pursued further.
[1020] In vitro, a site-specific transglutaminase linker performed significantly better than a lysine conjugation on a molar ratio basis in differentiated C2C12 mouse muscle cells for mediating exon skipping. Future testing of a high DAR (4) transglutaminase conjugate with a disulfide cleavable linker will be evaluated. [1021] Without wishing to be bound by theory, it is thought that, given a relative fast half- life of the 3E10 antibody, a more readily releasable linker, i.e., less hindered, may facilitate more rapid release of the PMO to mediate greater exon skipping in vivo. This design shows promise in vitro and may be a more optimal AOC design, e.g. SPDB or SPDP linker (Disulfide) (Figure 52).
[1022] AOCs having ASOs with the sequence of SEQ ID NO: 1100 (Figure 15) and the following linker designs were evaluated for exon skipping efficacy in in vitro in mouse C2C12 muscle cells and quantified for exon skipping after 4 days incubation:
• (Ly s)-DBCO-PEG8-PMO (D AR 3.8)
• (Lys)-DBCO-PEG4-SPDMV-PMO (DAR 3.4)
• (Lys)-DBCO-PEG8-PO4-PAB-PMO (DAR 3.3)
• (Tg)-DBCO-PEG8-PMO (DAR 2.0)
• (Tg)-DBCO-PEG4-SPDMV-PMO (DAR 1.7)
• (Tg)-CathB-PEG8-PMO (DAR 2.0)
[1023] Exon skipping of DMD preRNA was determined and quantified by Q-PCR. As shown in Figures 53A and 53B, titrations of the AOC conjugates demonstrated superior skipping activity with transglutaminase conjugation. In addition, AOCs comprising hindered disulfide SPDMV and phosphate cleavable linkers had similar potency and were superior to the noncleavable linker. The AOC comprising the peptide (Cathepsin B) cleavable linker was less potent than the AOC comprising the disulfide cleavable linker.
As shown in Figures 54A-54C, feasibility of conjugation and function were established for site- specific DAR 4 3E10 AOC transglutaminase conjugate comprising a hindered disulfide SPDMV linker (Tg-SPDP). Figure 54A shows a schematic of the Tg-SPDP 3E10 AOC. Figure 54B shows that a high degree of purity was observed for the Tg-SPDP 3E10 AOC. Figure 54C shows that the Tg-SPDP 3E10 AOC generated increased levels of exon skipping compared to a noncleavable 3E10 AOC (Tg-Peg8), or to two cleavable 3E10 AOCs (Tg-SPDMV and Tg-CathB). These results show that the Tg-SPDP 3E10 AOC was feasible and delivered superior potency. Sequences

Claims

1. A conjugate of Formula (I):
A-(L-Pr)q
Formula (I), wherein in Formula (I):
A is an antibody, antigen-binding fragment thereof or antigen-binding fragment thereof comprising a heavy chain variable region (VH) CDR1 comprising the amino acid sequence of SEQ ID NO:58, CDR2 comprising the amino acid sequence of SEQ ID NO:59, CDR3 comprising SEQ ID NO:60; and a light chain variable region (VL) CDR1 comprising the amino acid sequence of SEQ ID NO:61, CDR2 comprising the amino acid sequence of SEQ ID NO:62, CDR3 comprising the amino acid sequence of SEQ ID NO:63;
L is a linker;
P is an oligonucleotide capable of hybridizing to a pre-mRNA transcript, wherein the oligonucleotide induces exon skipping in the pre-mRNA transcript; r is an integer from 1 to 4; and q is an integer from 1 to 16.
2. The conjugate of claim 1, wherein the oligonucleotide hybridizes to an acceptor splice site, a donor splice site, or an exonic splice enhancer element of the pre-mRNA transcript.
3. The conjugate of claim 1 or 2, wherein the linker L comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heteroalkylene, optionally substituted heteroarylene, optionally substituted heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -S(O)-, -S(O)2-, -OP(O)ORa-, -OP(O)ORaO-, -P(O)ORaO-, -O-, -CRb 2-, -[(CRb2)1-120]1-50-, -C(O)-, -C(S)-, -C(=N-OH)-, -C(NRa)-, -C(NH2Cl)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -C(O)NRaSO2-, -SO2NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, -S(O)tN(Ra)- (where t is 1 or 2), -N(Ra)S(O)t- (where t is 1 or 2), and -XAA-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted alkenyl, optionally substituted fluoroalkenyl, optionally substituted cycloalkenyl, optionally substituted cycloalkenylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -C(0)NRa 2, -CO2Ra, -NRa 2, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted alkenyl, optionally substituted fluoroalkenyl, optionally substituted cycloalkenyl, optionally substituted cycloalkenylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; two independent Rb groups or an Ra and an Rb can be joined together to form an optionally substituted cycle; and
-XAA- is an amino acid sequence comprising 1 to 6 amino acid moieties.
4. The conjugate of claim 3, wherein each amino acid moiety of -XAA- is independently selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Vai), citrulline (Cit), and homocitrulline (HoCit).
5. The conjugate of any one of claims 1 to 4, wherein the linker L comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CR — CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, -[CH2O]1-18-, -[CH2CH2O]1-18-, -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, and -XAA-.
6. The conjugate of any one of claims 1 to 5, wherein the linker L comprises one or more groups selected from optionally substituted C1-C16 alkylene, -C=C-, -CR — CRa-, optionally substituted phenylene, optionally substituted C3-C6 cycloalkylene, -[CH2CH2O]1-16-, -[CH2CH2CH2O] 1-16-, optionally substituted 5- to 6-membered heteroarylene, optionally substituted 5- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb 2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)NRa-, -NRaC(O)O-, and -XAA-.
7. The conjugate of any one of claims 1 to 6, wherein the linker L comprises one or more groups selected from -[C(Rb)2]1-16-, -C=C-, -CRa= CRa-, -[CH2CH2O]1-16-, -NRa-, -N=CRa-, -CRa=N-. -S-, -OP(O)ORaO-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-,
X1, X2, and X3 are independently selected at each occurrence from NRa, N, CRb, S, and O.
8. The conjugate of any one of claims 1 to 7, wherein the linker L is of Formula (L- 1 ): Formula (L-l), wherein in Formula (L-l):
LA is a connecting moiety through which A is covalently attached to L'; L' is a bond or comprises one or more groups selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heteroalkylene, optionally substituted heteroarylene, optionally substituted heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -S(O)-, -S(O)2-, -OP(O)ORaO-, -O-, -CRb2-, -[(CRb 2)1-12O]1-50-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2C1)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -C(O)NRaSO2-, -SO2NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, -S(O)tN(Ra)-, -N(Ra)S(O)t-, and -XAA-; and
Lp is a connecting moiety through which P is covalently attached to L'.
9. The conjugate of any one of claims 1 to 8, wherein the linker L comprises at least one cleavable moiety.
10. The conjugate of claim 9, wherein the cleavable moiety comprises an acid-labile moiety, a reducibly-labile moiety, or an enzymatically-labile moiety.
11. The conjugate of claim 9 or claim 10, wherein the cleavable moiety comprises one or more groups selected from: wherein: each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl.
12. The conjugate of any one of claims 10 to 11, wherein the cleavable moiety comprises the reducibly-labile moiety -S-S-.
13. The conjugate of any one of claims 1 to 9, wherein the linker L is of Formula (L-10):
Formula (L-10), wherein in Formula (L-10):
LA is selected from a bond, -NRa'-, and -S-;
L1 is a bond or comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CR — CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C.3-C20 cycloalkylene, -[CH2O]1-18-, -[CH2CH2O]1-18-, -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb 2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, and -XAA-;
Lc is selected from an acid-labile moiety, a reducibly-labile moiety, and an enzymatically- labile moiety;
L2 is a bond or comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CR — CR1-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene. -[CH2O]1-18-, -[CH2CH2O]1-18-. -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -CRb 2-, -C(O)-. -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -OC(O)S-, -SC(O)O-, -OC(O)NRa-, -NRaC(O)O-, -SC(O)NRa-, -NRaC(O)S-, and -XAA-;
LP is selected from a bond, -NRa-, -S-, and -O-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -C(0)NRa 2, -CO2Ra, -NRa 2, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycloalkyl, or optionally substituted heteroaryl; and
-XAA- is an amino acid sequence comprising 1 to 4 amino acid moieties.
14. The conjugate of claim 12, wherein Lc is selected from: wherein: each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl.
15. The conjugate of claim 13 or claim 14, wherein Lc is -S-S-.
16. The conjugate of any one of claims 1 to 8, wherein the linker L is of Formula (L-l 1):
Formula (L-l 1), wherein in Formula (L-l 1):
LA is selected from a bond, -NH-, and -S-;
L1 is a bond or comprises one or more groups selected from -[C(Rb)2]1-16-, -C=C-, -CRa=CRa-, -[CH2CH2O]1-16-, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-, -NRaC(O)O-,
L2 is a bond or comprises one or more groups selected from -[C(Rb)2]1-16-, -C=C-,
-CRa=CRa-, -[CH2CH2O]1-16-, -NRa-, -N=CRa-, -CRa=N-, -S-, -OP(O)ORaO-, -O-, -C(0)-,
-C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-, -NRaC(O)O-,
LP is selected from a bond, -NRa -, and -O-; each R1 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or both Ri groups are taken together to form optionally substituted cycloalkyl; each R2 is independently selected from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or both R2 groups are taken together to form optionally substituted cycloalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each Ra is independently selected at each occurrence from hydrogen and optionally substituted alkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl; and
-XAA- is an amino acid sequence comprising 2 to 4 amino acid moieties.
17. The conjugate of any one of claims 1 to 8, wherein the linker L is of Formula (L-12): L2’ comprises one or more groups selected from -[C(Rb)2]1-10-, -[CH2CH2O]1-10-, -NRa-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)NRa-, -NRaC(O)-, -OC(O)O-, -XAA-, -OC(O)NRa-,
Lp is selected from a bond and -NRa-; each Ri is independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or both Ri groups are taken together to form optionally substituted C3-C6 cycloalkyl; each R2 is independently selected from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or both R2 groups are taken together to form optionally substituted C3-C6 cycloalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 fluoroalkyl, and optionally substituted C3-C6 cycloalkyl; each Ra is independently selected at each occurrence from hydrogen and optionally substituted C1-C6 alkyl; each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 fluoroalkyl, and optionally substituted C3-C6 cycloalkyl; or two independent Rb groups attached to the same carbon atom are taken together to form optionally substituted C3-C6 cycloalkyl; and
-XAA- is an amino acid sequence comprising 2 or 3 amino acid moieties.
18. The conjugate of claim 16 or claim 17, wherein at least one Ri or R2 is other than hydrogen.
19. The conjugate of any one of claims 16 to 18, wherein at least one Ri is an optionally substituted C1-C8 alkyl.
20. The conjugate of any one of claims 16 to 18, wherein each Ri is independently an optionally substituted C1-C8 alkyl.
21. The conjugate of any one of claims 16 to 20, wherein at least one R2 is an optionally substituted C1-C8 alkyl.
22. The conjugate of any one of claims 16 to 20, wherein each R2 is independently an optionally substituted C1-C8 alkyl.
23. The conjugate of any one of claims 1 to 11, wherein the linker L is selected from:
24. The conjugate of any one of claims 1 to 17, wherein the linker L is selected from:
25. The conjugate of any one of claims 9 to 24, wherein the linker is a cleavable linker.
26. The conjugate of claim 25, wherein the cleavable linker is a cathepsin-L substrate.
27. The conjugate of claim 26, wherein the linker comprises a dipeptide selected from a -Phe-
Gln- dipeptide, a -Val-Gln- dipeptide, a -Leu-Gin- dipeptide, a -Tyr-Met- dipeptide, a -Phe-Arg- dipeptide, a -Phe-Gly- dipeptide, a -Trp-Thr- dipeptide, a -Tyr-Gly- dipeptide, a -Phe-Thr- dipeptide, and a -Val-Gly- dipeptide.
28. The conjugate of claim 26, wherein the linker comprises a -Phe-Gln- dipeptide, a -Val- Gln- dipeptide, a -Leu-Gin- dipeptide, or a -Tyr-Met- dipeptide.
29. The conjugate of claim 26, wherein the linker comprises a -Phe-Gln- dipeptide.
30. The conjugate of claim 26, wherein the linker comprises a -Val-Gln- dipeptide.
31. The conjugate of claim 26, wherein the linker comprises a -Leu-Gin- dipeptide.
32. The conjugate of claim 26, wherein the linker comprises a -Tyr-Met- dipeptide.
33. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a lysine of the 3E10 antibody or antigen-binding fragment thereof.
34. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a cysteine of the 3E10 antibody or antigen-binding fragment thereof.
35. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a histidine of the 3E10 antibody or antigen-binding fragment thereof.
36. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to an arginine of the 3E10 antibody or antigen-binding fragment thereof.
37. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to an aspartic acid of the 3E10 antibody or antigen-binding fragment thereof.
38. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a glutamine of the 3E10 antibody or antigen-binding fragment thereof.
39. The conjugate of claim 38, wherein the conjugate has a drug to antibody ratio (DAR) of at least 4: 1.
40. The conjugate of claim 38 or 39, wherein the linker is a branched linker attached to at least two copies of the oligonucleotide moiety.
41. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a serine of the 3E10 antibody or antigen-binding fragment thereof.
42. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a threonine of the 3E10 antibody or antigen-binding fragment thereof.
43. The conjugate of any one of claims 25-32, wherein the cleavable linker is conjugated to a tyrosine of the 3E10 antibody or antigen-binding fragment thereof.
44. The conjugate of any one of claims 1 to 8, wherein the linker L is of Formula (L-20):
Formula (L-20), wherein in Formula (L-20):
LA is selected from a bond, -NRa'-, and -S-;
L3 is a bond or comprises one or more groups selected from -[C(Rb)2]1-8-, -NRa-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2C1)-, -C=C-, -CRa= CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, optionally substituted 5- to 18-membered heteroarylene, and optionally substituted 3- to 20-membered heterocycloalkylene;
Lx comprises one or more groups selected from optionally substituted C1-C18 alkylene, -C=C-, -CR — CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, -[CH2O]1-18-, -[CH2CH2O] 1-18-, -[CH2CH2CH2O]1-18-, optionally substituted 5- to 18-membered heteroarylene, optionally substituted 3- to 20-membered heterocycloalkylene, -NRa-, -S-, -O-, -CRb 2-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2C1)-, -C(O)O-, -OC(O)-, -C(O)S-, -SC(O)-, -C(O)NRa-, and -NRaC(O)-;
L4 is a bond or comprises one or more groups selected from -[C(Rb)2]i-8-, -NRa-, -C(O)-, -C(S)-, -C(NRa)-, -C(NH2Cl)-, -C=C-, -CR — CRa-, optionally substituted 6- to 14-membered arylene, optionally substituted C3-C20 cycloalkylene, optionally substituted 5- to 18-membered heteroarylene, and optionally substituted 3- to 20-membered heterocycloalkylene;
Lp is selected from a bond, -NRa -, -S-, and -O-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each Ra is independently selected at each occurrence from hydrogen, optionally substituted alkyl, and optionally substituted heteroalkyl; and each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -C(O)NRa 2, -CO2Ra, -NRa 2, optionally substituted alkyl, optionally substituted fluoroalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkylalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycloalkyl, or optionally substituted heteroaryl.
45. The conjugate of any one of claims 1 to 8, wherein the linker L is of Formula (L-21):
Formula (L-21), wherein in Formula (L-21):
LA is selected from a bond and -NH-;
Lx comprises one or more groups selected from optionally substituted -[C(Rb)2]1-16-, -C=C-, -CR — CRa-, -[CH2CH2CH2O]1-16-, -NRa-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)S-,
Lp is selected from a bond and -NRa-; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each Ra' is independently selected at each occurrence from hydrogen and optionally substituted C1-C6 alkyl; and each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl.
46. The conjugate of any one of claims 1 to 8, wherein the linker L is of Formula (L-22a) or Formula (L-22b): wherein in Formulas (L-22a) and (L-22b):
LA is selected from a bond and -NH-;
Lx comprises one or more groups selected from optionally substituted -[C(Rb)2] 1-10-,
-C=C-, -CR:— CRa-, -[CH2CH2CH2O]1-10-, -NRa-, -C(0)-, -C(0)0-, -OC(O)-, -C(O)S-, -SC(O)-,
Lp is selected from a bond and -NRa -; each Ra is independently selected at each occurrence from hydrogen, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; each Ra is independently selected at each occurrence from hydrogen and optionally substituted C1-C6 alkyl; and each Rb is independently selected at each occurrence from hydrogen, halide, -OH, -SO3H, -OPO3H2, -PO3H2, -CO2Ra, -NRa 2, optionally substituted C1-C8 alkyl, optionally substituted C1-C8 fluoroalkyl, optionally substituted C3-C6 cycloalkyl, optionally substituted phenyl, optionally substituted benzyl, optionally substituted 5- to 10-membered heterocycloalkyl, optionally substituted 5- to 6-membered heteroaryl; or two independent Rb groups are taken together to form optionally substituted cycloalkyl.
47. The conjugate of any one of claims 1 to 8, wherein the linker L is selected from:
48. The conjugate of any one of claims 44 to 47, wherein the linker is a non-cleavable linker.
49. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a lysine of the 3E10 antibody or antigen-binding fragment thereof.
50. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a cysteine of the 3E10 antibody or antigen-binding fragment thereof.
51. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a histidine of the 3E10 antibody or antigen-binding fragment thereof.
52. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to an arginine of the 3E10 antibody or antigen-binding fragment thereof.
53. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to an aspartic acid of the 3E10 antibody or antigen-binding fragment thereof.
54. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a glutamine of the 3E10 antibody or antigen -binding fragment thereof.
55. The conjugate of claim 54, wherein the conjugate has a drug to antibody ratio (DAR) of at least 4: 1.
56. The conjugate of claim 54 or 55, wherein the linker is a branched linker attached to at least two copies of the oligonucleotide moiety.
57. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a serine of the 3E10 antibody or antigen-binding fragment thereof.
58. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a threonine of the 3E10 antibody or antigen-binding fragment thereof.
59. The conjugate of claim 48, wherein the non-cleavable linker is conjugated to a tyrosine of the 3E10 antibody or antigen-binding fragment thereof.
61. The conjugate of any one of claims 1 to 17, wherein each amino acid moiety of -XAA- is independently selected from alanine (Ala), arginine (Arg), glycine (Gly), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), valine (Vai), citrulline (Cit), and homocitrulline (HoCit).
62. The conjugate of any one of claims 1 to 17, wherein each amino acid moiety of -XAA- is independently selected from alanine (Ala), glycine (Gly), lysine (Lys), phenylalanine (Phe), valine (Vai), and citrulline (Cit).
63. The conjugate of any one of claims 1 to 16, wherein the amino acid sequence -XAA- is selected from -Val-Cit-, -Cit-Val-, -Vai-Ala-, -Ala-Vai-, -Phe-Lys-, -Lys-Phe-, -Ala-Ala-, -Val-Val-, -Gly-Gly-, -Ala-Ala-Ala-, -Gly-Gly-Gly-, -Gly-Gly-Phe-Gly-(SEQ ID NO: 1032), -Gly-Phe-Gly-Gly-(SEQ ID NO: 1033),
-Gly-Gly-Gly-Phe-(SEQ ID NO: 1034), -Phe-Gly-Gly-Gly-(SEQ ID NO: 1035), and -Gly-Gly-Gly-Gly-(SEQ ID NO: 1036).
64. The conjugate of any one of claims 1 to 17, wherein the amino acid sequence -XAA- is selected from -Val-Cit-, -Cit-Val-, -Val-Ala-, -Ala-Vai-, -Phe-Lys-, -Lys-Phe-, -Ala-Ala-, -Val-Val-, -Gly-Gly-, -Ala-Ala-Ala-, and -Gly-Gly-Gly-.
65. The conjugate of any one of claims 1 to 63, wherein the single stranded oligonucleotide P is a phosphorodiamidate morpholino oligonucleotide or an antisense oligonucleotide.
66. The conjugate of any one of claims 1 to 65, wherein the single stranded oligonucleotide P is delivered into a muscle cell.
67. The conjugate of any one of claims 1 to 66, wherein the single stranded oligonucleotide P induces skipping of exon 23 of the DMD gene.
68. The conjugate of claim 65, wherein the phosphorodiamidate morpholino oligonucleotide comprises the sequence 5'-C6 Amino-GGCCAAACCTCGGCTTACCTGAAAT-3' (SEQ ID NO:408).
69. The conjugate of claim 65, wherein the antisense oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 1045, SEQ ID NOs: 158-222, SEQ ID NO:395- 405, and SEQ ID NO:410-988.
70. The conjugate of claim 65, wherein the antisense oligonucleotide is a peptide nucleic acid (PNA) oligonucleotide.
71. The conjugate of claim 70, wherein the peptide nucleic acid (PNA) oligonucleotide comprises the sequence (C)-3’-TAAAGTCCATTCGGCTCCAAACCGG-C6 Amino-5’(N) (SEQ ID NO: 409).
72. The conjugate of any one of claims 1 to 71, wherein the single stranded oligonucleotide P comprises at least from about 10 to about 30 nucleotides in length.
73. The conjugate of claim 1, wherein the truncated protein modulates muscular dystrophy.
74. The conjugate of claim 73, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.
75. The conjugate of any one of claims 1 to 74, wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO:9, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, CDR3 comprises SEQ ID NO: 11; and the VH CDR1 comprises the amino acid sequence of SEQ ID NO:15, CDR2 comprises the amino acid sequence of SEQ ID NO:4, CDR3 comprises the amino acid sequence of SEQ ID NO:5.
76. The conjugate of any one of claims 1 to 74, wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO:29, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, CDR3 comprises SEQ ID NO: 11; and the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 15, CDR2 comprises the amino acid sequence of SEQ ID NO:26, CDR3 comprises the amino acid sequence of SEQ ID NO:5.
77. The conjugate of any one of claims 1 to 74, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO:21 and a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 14.
78. The conjugate of any one of claims 1 to 74, wherein the antibody or antigen-binding fragment thereof comprises a full length light chain (LC) comprising an amino acid sequence of SEQ ID NO:20 and a full length heavy chain (HC) comprising an amino acid sequence of SEQ ID NO:13.
79. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VL-H1 (SEQ ID NO:85), 3E10-VL-H2 (SEQ ID NO:86), 3E10-VL-H3 (SEQ ID NO:87), 3E10-VL-H4 (SEQ ID NO:88), 3E10-VL-H5 (SEQ ID NO:89), and 3E10-VL-H6 (SEQ ID NO:90); and a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of 3E10-VH-H1 (SEQ ID NO:64), 3E10-VH-H2 (SEQ ID NO:65), 3E10-VH-H3 (SEQ ID NO:66), 3E10-VH-H4 (SEQ ID NO:67), 3E1O-VH-H5 (SEQ ID NO:68), 3E10-VH-H6 (SEQ ID NO:69), and 3E10- VH-H7 (SEQ ID NO:70).
80. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence selected from the group consisting of 3E10-VL-H1 (SEQ ID NO:85), 3E10-VL-H2 (SEQ ID NO:86), 3E10-VL- H3 (SEQ ID NO:87), 3E10-VL-H4 (SEQ ID NO:88), 3E10-VL-H5 (SEQ ID NO:89), and 3E10- VL-H6 (SEQ ID NOVO); and a heavy chain variable domain (VH) comprising an amino acid sequence selected from the group consisting of 3E10-VH-H1 (SEQ ID NO:64), 3E10-VH-H2 (SEQ ID NO:65), 3E10-VH- H3 (SEQ ID NO:66), 3E10-VH-H4 (SEQ ID NO:67), 3E10-VH-H5 (SEQ ID NO:68), 3E10- VH-H6 (SEQ ID NO:69), and 3E10-VH-H7 (SEQ ID NO:70).
81. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises a VL / VH pair selected from the group consisting of: (a) VL-hl (SEQ ID NO:85) and VH-hl (SEQ ID NO:64),
(b) VL-hl (SEQ ID NO:85) and VH-h2 (SEQ ID NO:65),
(c) VL-hl (SEQ ID NO:85) and VH-h3 (SEQ ID NO:66),
(d) VL-hl (SEQ ID NO:85) and VH-h4 (SEQ ID NO:67),
(e) VL-h2 (SEQ ID NO:86) and VH-hl (SEQ ID NO:64),
(f) VL-h2 (SEQ ID NO:86) and VH-h2 (SEQ ID NO:65),
(g) VL-h2 (SEQ ID NO:86) and VH-h3 (SEQ ID NO:66),
(h) VL-h2 (SEQ ID NO:86) and VH-h4 (SEQ ID NO:67),
(i) VL-h3 (SEQ ID NO: 87) and VH-hl (SEQ ID NO: 64),
(j) VL-h3 (SEQ ID NO: 87) and VH-h2 (SEQ ID NO: 65),
(k) VL-h3 (SEQ ID NO:87) and VH-h3 (SEQ ID NO:66),
(l) VL-h3 (SEQ ID NO: 87) and VH-h4 (SEQ ID NO: 67),
(m) VL-h4 (SEQ ID NO: 88) and VH-hl (SEQ ID NO: 64),
(n) VL-h4 (SEQ ID NO:88) and VH-h2 (SEQ ID NO:65),
(o) VL-h4 (SEQ ID NO:88) and VH-h3 (SEQ ID NO:66),
(p) VL-h4 (SEQ ID NO:88) and VH-h4 (SEQ ID NO:67),
(q) VL-h5 (SEQ ID NO:89) and VH-h5 (SEQ ID NO:68),
(r) VL-h5 (SEQ ID NO:89) and VH-h6 (SEQ ID NO:69),
(s) VL-h6 (SEQ ID NO:90) and VH-h5 (SEQ ID NO:68), and
(t) VL-h6 (SEQ ID NO:90) and VH-h6 (SEQ ID NO:69).
82. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) comprising an amino acid sequence that is at least 95% identical to 3E10-VL-H6 (SEQ ID NO:90) and a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 95% identical to 3E10-VH-H6 (SEQ ID NO:69).
83. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) comprising the amino acid sequence of 3E10-VL-H6 (SEQ ID NO:90) and a heavy chain variable domain (VH) comprising the amino acid sequence of 3E10-VH-H6 (SEQ ID NO:69).
84. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 105 and a heavy chain variable domain (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 117.
85. The conjugate of any one of claims 1 to 78, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 105 and a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 117.
86. A method for treating a genetic disease or disorder in a subj ect in need thereof, the method comprising administering a therapeutically effective amount of a conjugate according to any one of claims 1 to 85 to the subject.
87. The method of claim 86, wherein the disease or disorder is a neurogenetic disease, a musculoskeletal disorder, a cardiovascular disease, a metabolic disease, a cancer, a lung disorder, or a disease that can be benefitted by exon-skipping therapies.
88. The method of claim 87, wherein the neurogenetic disease is CDG, FD, PBD1A, MRD23, EPM5, FHM1, AHC, AGS6, EIEEF4, VWM, ICCA, BFIE, EKD1, ALGS, TSC, USH, PTHS, SMS, GEFSP9, HGPS, FTDP-17, PHMDS, SCZD15, NF2, or PARK8.
89. The method of claim 87, wherein the musculoskeletal disorder is DMD, DM1, DM2, SMA, or IBMPFDl.
90. The method of claim 87, wherein the cardiovascular disease is FH.
91. The method of claim 87, wherein the metabolic disease is ACADMD, PKU, or NPC1.
92. The method of claim 87, wherein the cancer is a RAS-associated cancer or MLD.
93. The method of claim 87, wherein the lung disorder is CF.
94. The method of claim 87, wherein the disease that can be benefitted by exon-skipping therapies is IBS or DEB.
95. A method of treating Duchenne muscular dystrophy (DMD) in a subject in need thereof, the method comprising administering a therapeutically effective amount of a conjugate according to any one of claims 1 to 85 to the subject.
96. A conjugate according to any one of claims 1 to 85 for use in the treatment of a genetic disease or disorder.
97. The conjugate of claim 96, wherein the disease or disorder is a neurogenetic disease, a musculoskeletal disorder, a cardiovascular disease, a metabolic disease, a cancer, a lung disorder, or a disease that can be benefitted by exon-skipping therapies.
98. The conjugate of claim 96, wherein the neurogenetic disease is CDG, FD, PBD1A, MRD23, EPM5, FHM1, AHC, AGS6, EIEEF4, VWM, ICCA, BFIE, EKD1, ALGS, TSC, USH, PTHS, SMS, GEFSP9, HGPS, FTDP-17, PHMDS, SCZD15, NF2, or PARK8.
99. The conjugate of claim 96, wherein the musculoskeletal disorder is DMD, DM1, DM2, SMA, or IBMPFDl.
100. The conjugate of claim 96, wherein the cardiovascular disease is FH.
101. The conjugate of claim 96, wherein the metabolic disease is ACADMD, PKU, or NPC1.
102. The conjugate of claim 96, wherein the cancer is a RAS-associated cancer or MLD.
103. The conjugate of claim 96, wherein the lung disorder is CF.
104. The conjugate of claim 96, wherein the disease that can be benefitted by exon-skipping therapies is IBS or DEB.
105. A conjugate according to any one of claims 1 to 85 for use in the treatment of Duchenne muscular dystrophy (DMD).
EP24718015.1A 2023-03-23 2024-03-25 Compositions and methods for delivering antibody oligonucleotide conjugates for exon skipping Pending EP4683674A1 (en)

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