CN118043078A - SIRP-alpha antibodies and conjugates - Google Patents

SIRP-alpha antibodies and conjugates Download PDF

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CN118043078A
CN118043078A CN202280067118.9A CN202280067118A CN118043078A CN 118043078 A CN118043078 A CN 118043078A CN 202280067118 A CN202280067118 A CN 202280067118A CN 118043078 A CN118043078 A CN 118043078A
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seq
antibody
amino acid
acid sequence
conjugate
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李敏
O·哈拉比
A·陈
E·R·桑嘉朗
T·C-C·郭
B·J·希姆
H·I·万
J·庞斯
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Tarak Therapy
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Tarak Therapy
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Priority claimed from PCT/US2022/075381 external-priority patent/WO2023028511A1/en
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Abstract

The present disclosure provides anti-SIRP-a polypeptide antibodies and oligonucleotide conjugates thereof. Also provided are methods of their associated preparation and methods of use, including therapeutic uses.

Description

SIRP-alpha antibodies and conjugates
Cross reference to related applications
The present application claims priority from U.S. provisional application No. 63/236,989 filed on 25 th 8 of 2021 and U.S. provisional application No. 63/256,270 filed on 15 th 10 of 2021, each of which is hereby incorporated by reference in its entirety.
Reference to an electronic sequence Listing
The contents of the electronic sequence Listing (186492000440 seqlist. Xml; size: 315,160 bytes; and date of creation: 2022, month 8, 22) are incorporated herein by reference in their entirety.
Technical Field
The present disclosure relates to anti-SIRP-a antibodies and conjugates thereof, and uses thereof, including therapeutic uses.
Background
Signal regulatory protein alpha (SIRP-alpha) is part of a family of cell surface receptors that play a critical role in the regulation of the immune system (see, e.g., barclay, A.N. and Brown, M.H. (2006) Nat. Rev. Immunol. 6:457-64). One of the major effects of SIRP-a is that it modulates immune responses via interaction with CD 47. CD47 is expressed on the surface of various cell types. When the IgSF domain of CD47 binds to the extracellular domain (e.g., D1 domain) of SIRP-a expressed on immune cells (e.g., macrophages), this transduces SIRP-a mediated signals in immune cells, preventing phagocytosis of cells expressing CD 47. Thus, CD47 serves to transmit a signal called "don't me" to the immune system to prevent phagocytosis of healthy cells (see e.g. WO2015/138600 and Weiskopf, k. Et al (2013) Science 341:88-91). However, CD47 is also shown to be highly expressed by various cancers, and its interaction with SIRP-alpha in this case is thought to allow tumors to mimic healthy "do-it-yourself" signals in order to circumvent immune surveillance and phagocytosis by macrophages (see, e.g., majeti, R.et al (2009) Cell 138:286-99; zhao, X.W. et al (2011) Proc.Natl. Acad. Sci.108: 18342-7). Thus, antibodies that block this interaction are highly desirable.
SIRP- α is expressed on the surface of a variety of cells, including leukocytes such as dendritic cells, eosinophils, neutrophils and macrophages. SIRP- α includes extracellular domains that interact with external stimuli such as ligands and intracellular domains that mediate various intracellular signals. SIRP- α is a myelosuppressive receptor that inhibits immune activation after binding to its ligand CD 47. Blocking the CD 47-sirpa bone marrow checkpoint pathway has been shown to promote bone marrow-mediated antitumor function, thereby inducing adaptive immunity (Kuo, t.et al (2020) j.hemalol.oncol.13:160). In addition, SIRPalpha is highly expressed in a variety of tumor types, including renal cell carcinoma and melanoma (Yanagita, T.et al (2017) JCI Insight 2:e89140).
Pathogen-associated molecular patterns (PAMPs) are molecules associated with a variety of pathogens and are recognized by toll-like receptors (TLRs) and other Pattern Recognition Receptors (PRRs) that activate the innate immune response. The ability of PAMPs to recruit the immune system in the absence of pathogens provides strategies (e.g., anti-cancer therapies) that involve the treatment of a variety of diseases through cellular destruction using the innate immune system response. One class of PAMPs that has been investigated for a variety of therapeutic applications is immunostimulatory oligonucleotides, such as oligodeoxynucleotides (CpG ODNs) containing unmethylated cytosine-guanine dinucleotides (CpG) (e.g., attomode (agatolimod)). CpG ODNs are believed to mediate TLR9 dimerization in immune cells (e.g., B cells, monocytes and plasmacytoid dendritic cells (pdcs)) to up-regulate cytokines (e.g., type I interferons and interleukins), thereby activating natural killer cells.
Toll-like receptor 9 (TLR 9), also known as CD289, is an important receptor expressed in cells of the immune system including Dendritic Cells (DCs), B lymphocytes, macrophages, natural killer cells and other antigen presenting cells. TLR9 activation triggers an intracellular signaling cascade, leading to activation, maturation, proliferation and cytokine production of these immune cells, thus bridging innate and adaptive immunity. Martinez-Campos et al, visual immunol.2016,30,98-105; notley et al, sci.rep.2017,7,42204. Natural TLR-9 agonists include oligodeoxynucleotides (CpG ODNs) containing unmethylated cytosine-guanine dinucleotides (cpgs).
CpG ODNs can include, for example, oligodeoxynucleotides having poly-G tails and phosphorothioate backbones at the 3 'and 5' ends, including the central palindromic sequence of the phosphate backbone with CpG within its central palindromic sequence; or an oligodeoxynucleotide having a complete phosphorothioate backbone and a sequence for TLR9 activation at the 5' end; or an oligodeoxynucleotide having a complete phosphorothioate backbone and a 3' end sequence capable of duplex formation. However, cpG ODNs are generally prone to degradation in serum, and thus the pharmacokinetics of CpG ODNs may be one of the limiting factors in their development as therapeutic agents. Furthermore, cpG ODNs often exhibit an uneven tissue distribution in vivo, with the major sites of accumulation in the liver, kidneys and spleen. Such a profile may trigger off-target activity and localized toxicity associated with PAMPs.
All references, including patent applications, patent publications, and scientific literature, cited herein are hereby incorporated by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference.
Disclosure of Invention
As demonstrated herein, conjugates were found to integrate TLR9 activation and blocking of CD 47-SIRP-a interaction with bone marrow cells such that the anti-tumor immune response engages both the innate and adaptive immune systems. Without wishing to be bound by theory, it is believed that anti-SIRP-a antibody-CpG oligonucleotide conjugates may have the additional benefit of preferential tumor cell targeting in SIRP-a expressing cancers.
In one aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment is linked to one or more Q tag peptides (Q) comprising at least one glutamine residue, and wherein each immunomodulatory oligonucleotide is linked to a Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
Wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61);
Wherein each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection to the joint L;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
U 5' is-H or halogen;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer from 0 to 2.
In another aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPA SVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof
Each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection with the joint L.
In another aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDT YYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof
Each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection with the joint L.
In another aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide (P) is linked to a Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof.
In another aspect, provided herein is a conjugate comprising an antibody or antigen binding fragment (Ab) thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide, wherein the antibody comprises: two antibody light chains each comprising a light chain Variable (VL) domain comprising a CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), a CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and a CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); two antibody heavy chains each comprising a heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57), and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58); and two Q tag peptides comprising peptide sequence RPQGF (SEQ ID NO: 47); wherein the Q tag peptides are each linked to the C-terminus of one of the antibody heavy chains; and wherein at least one of the Q tag peptides is linked to an immunomodulatory oligonucleotide (P) via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide, as shown in fig. 9A or 9B.
In another aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment (Ab) comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q), wherein each immunomodulatory oligonucleotide (P) is linked to a Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof; and wherein the antibody heavy chain comprises an Fc region (e.g., a human IgG1, igG2, or IgG4 Fc region) comprising an N297A substitution, the amino acid positions being numbered according to the EU index. In another aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue;
Wherein each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection to the joint L; and
Wherein the antibody heavy chain comprises an Fc region (e.g., a human IgG1, igG2, or IgG4 Fc region) comprising an N297A substitution, the amino acid positions being numbered according to the EU index. In another aspect, provided herein is a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue;
Wherein each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection to the joint L; and
Wherein the antibody heavy chain comprises an Fc region (e.g., a human IgG1, igG2, or IgG4 Fc region) comprising an N297A substitution, the amino acid positions being numbered according to the EU index. In another aspect, provided herein is a conjugate comprising an antibody or antigen binding fragment (Ab) thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide, wherein the antibody comprises: two antibody light chains each comprising a light chain Variable (VL) domain comprising a CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), a CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and a CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); two antibody heavy chains each comprising a heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57), and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58); and two Q tag peptides; wherein the Q tag peptides are each linked to the C-terminus of or present in the Fc region of one of the antibody heavy chains; wherein the antibody heavy chain comprises an Fc region (e.g., a human IgG1, igG2, or IgG4 Fc region) comprising an N297A substitution, the amino acid positions being numbered according to the EU index; and wherein at least one of the Q tag peptides is linked to an immunomodulatory oligonucleotide (P) via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide, as shown in fig. 9A or 9B. In some embodiments, Q295 of the Fc region serves as the glutamine residue of the Q tag peptide.
In another aspect, provided herein is a conjugate comprising an antibody (Ab) that specifically binds to an extracellular domain of a human SIRP-a polypeptide, at least one Q tag peptide sequence comprising a glutamine residue, and at least one immunomodulatory oligonucleotide (P), wherein the antibody or antigen-binding fragment thereof (Ab) comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); wherein the Q tag peptide sequence is naturally occurring or synthetic; wherein each immunomodulatory oligonucleotide is linked to a Q tag via an amide bond to the glutamine residue and a linker (L); and wherein at least one Q tag peptide sequence is selected from the group consisting of SEQ ID NOS: 39-55.
In some embodiments according to any of the embodiments described herein, the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNA MSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62). In some embodiments, the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAP GKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62), and the VL domain comprises the amino acid sequence of SYELTQPPSVSVSPGQTA RITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGS SSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTQLTVL(SEQ ID NO:63). In some embodiments, the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAP GKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62), and the VL domain comprises the amino acid sequence of SYELTQPPSVSVSPGQT ARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSG SSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTKLTVL(SEQ ID NO:64). In some embodiments, the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAP GKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62), and the VL domain comprises the amino acid sequence of SYELTQPPSVSVSPGQTA RITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGS SSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTELTVL(SEQ ID NO:65).
In some embodiments according to any of the embodiments described herein, the antibody is a monoclonal antibody. In some embodiments, the antibody is a Fab, F (ab') 2, fab ' -SH, fv, or scFv antibody or antibody fragment. In some embodiments, the antibody is a humanized antibody, a human antibody, or a chimeric antibody or fragment thereof. In some embodiments, the antibody comprises an antibody heavy chain comprising a VH domain and an Fc region of the invention. In some embodiments, the antibody heavy chain comprises a human IgG1, human IgG2, or human IgG4 Fc region. In some embodiments, the antibody heavy chain comprises a human IgG1 Fc region comprising L234A, L a and/or G237A substitutions, the amino acid positions being numbered according to the EU index. In some embodiments, the antibody heavy chain comprises a wild-type human IgG1 Fc region. In some embodiments, the antibody heavy chain comprises a human IgG1 Fc region comprising the N297A substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the antibody heavy chain comprises a human IgG1 Fc region comprising a D265A substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the antibody heavy chain comprises a wild-type human IgG2 Fc region. In some embodiments, the antibody heavy chain comprises a human IgG2 Fc region comprising the N297A substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the antibody heavy chain comprises a human IgG4 Fc region comprising an S228P substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the Fc region comprises an N297A substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the conjugate further comprises an immunomodulatory oligonucleotide P linked to Q295 of the Fc region residue, as shown in the following formula:
wherein L is a linker moiety linked to Q295 of the Fc region via an amide bond.
In some embodiments according to any of the embodiments described herein, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide, the antibody heavy chain comprising amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVR QAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGRPQGFGPP(SEQ ID NO:66)., in some embodiments, the antibody comprises an antibody heavy chain comprising amino acid sequence EVQLVESGGGVV QPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGS DTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:87)., in some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:87 and a Q tag sequence of the disclosure. In some embodiments, the antibody comprises an antibody heavy chain comprising an amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP(SEQ ID NO:67)., in some embodiments, an antibody heavy chain comprising an amino acid sequence EVQLVESGGGVVQPGG SLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:88)., in some embodiments, an antibody heavy chain comprising an amino acid sequence comprising SEQ ID No. 88, and a Q tag sequence of the disclosure. In some embodiments, the antibody comprises an antibody heavy chain comprising an amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP(SEQ ID NO:68)., in some embodiments, an antibody heavy chain comprising an amino acid sequence EVQLVESGGGVVQPG GSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:89)., in some embodiments, an antibody heavy chain comprising an amino acid sequence comprising SEQ ID NO:89, and a Q tag sequence of the disclosure.
In some embodiments according to any of the embodiments described herein, the antibody comprises an antibody light chain comprising the VL domain and a light chain Constant (CL) domain comprising amino acid sequence GQPKANPTVTLFPP SSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS(SEQ ID NO:69)., in some embodiments, the antibody comprises an antibody light chain comprising the VL domain and a light chain Constant (CL) domain comprising amino acid sequence GQPKANPTVTLFPPSSEELQANKATLVC LISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSSDKYAASSYLS LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:70)., in some embodiments, the antibody comprises an antibody light chain comprising the VL domain and a light chain Constant (CL) domain comprising amino acid sequence GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS(SEQ ID NO:71)., in some embodiments, the antibody comprises an antibody light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 72-80. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 67 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, the antibody comprises an antibody heavy chain having a C-terminal Q tag peptide comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, the antibody comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, the antibody heavy and/or light chain further comprises a Q tag peptide sequence of the disclosure.
In some embodiments according to any of the embodiments described herein, each of the one or more Q tag peptides (Q) comprises a peptide sequence having 5 to 15 amino acid residues. In some embodiments, each of the one or more Q tag peptides is naturally occurring. In some embodiments, each of the one or more Q tag peptides comprises a sequence independently selected from the group consisting of SEQ ID NOs 39-55. In some embodiments, each of the one or more Q tag peptides comprises peptide sequence RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48), or RPQGFGPP (SEQ ID NO: 49). In some embodiments, each of the one or more Q-tag peptides comprises a peptide sequence RPQGF (SEQ ID NO: 47). In some embodiments, the at least one Q tag peptide sequence comprises peptide sequence RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48), or RPQGFGPP (SEQ ID NO: 49). In some embodiments, the at least one Q tag peptide sequence comprises peptide sequence RPQGFGPP (SEQ ID NO: 49). In some embodiments, the antibody comprises two antibody heavy chains and two antibody light chains, and wherein one or both heavy chains further comprise a Q tag. In some embodiments, the Q tag is fused to the C-terminus of one or both of the heavy chains. In some embodiments, the Q tag is within the Fc domain. In some embodiments, the antibody comprises two antibody heavy chains and two antibody light chains, and wherein one or both light chains further comprise a Q tag. In some embodiments, the conjugate induces activation of TLR 9. In some embodiments, 1 or 2Q tags are attached to the antibody. In some embodiments, the DAR of the conjugate is 1 or 2.
In some embodiments, the linker L comprises a polyethylene glycol moiety. In some embodiments, the linker L is
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to T 3, and/>Indicating the point of attachment to the remainder of the conjugate. In some embodiments, Z is S. In some embodiments, the oligonucleotide P comprises at least one pair of gem (geminal) T 1 and T 2, wherein T 1 is S and T 2 is S -. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 1. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 2. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 3. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 5. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 6. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 7. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 8. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 9. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 10. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 11. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 12. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 13. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 14. In some embodiments, the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 15. In some embodiments, the oligonucleotide P comprises at least two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S -. In some embodiments, R 5' is H. In some embodiments, R 5' is methoxy. In some embodiments, R c1 is H. In some embodiments, R c1 is methoxy. In some embodiments, R 2 is methyl. In some embodiments, R 2 is H. In some embodiments, U 5' is bromine. In some embodiments, U 5' is-H. In some embodiments, m is an integer from 20 to 25. In some embodiments, m is 24. In some embodiments, each P independently comprises an oligonucleotide selected from table 2, table 14, and table 15. In some embodiments, each linker (L) and each immunomodulatory oligonucleotide (P) independently comprises an oligonucleotide selected from table 2, table 16, and table 17. In some embodiments, each P independently comprises an oligonucleotide selected from the group consisting of SEQ ID NOS 1-38 and 129-166. In some embodiments, each immunomodulatory oligonucleotide P is independently
Wherein the method comprises the steps of
B and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5;
Indicating the point of attachment of the immunomodulatory oligonucleotide P to the remainder of the conjugate; x 5' is a inclusion structure/> A 5' terminal nucleoside of (2);
X 3' is a containing structure 3' Terminal nucleoside of (2);
y PTE is a containing structure Wherein is indicates the point of attachment to the rest of the oligonucleotide and/>Indicating the point of attachment to the linker L, or if L is not present,/>Indicating a point of attachment to the Q tag peptide at the glutamine residue via an amide bond;
y 3' is a containing structure Is a terminal phosphotriester of (a);
each X N is independently an inclusion structure A nucleoside of (2);
Each Y N is independently an inclusion structure Is a nucleoside linker; wherein B N is independently a modified or unmodified nucleobase;
Each R N is independently-H or-O-C 1-4 alkyl, wherein the C 1-4 alkyl of the-O-C 1-4 alkyl is optionally further substituted with-O-C 1-C4 alkyl;
b 5' and B 3' are independently modified or unmodified nucleobases;
R 5' and R 3' are independently-H or-O-C 1-C4 alkyl, wherein the C 1-4 alkyl of the-O-C 1-4 alkyl is optionally further substituted with-O-C 1-4 alkyl;
each T 1 is independently O or S;
Each T 2 is independently O - or S -; and
T 3 is a group comprising an oligoethylene glycol moiety; and
R 1 is C 1-4 alkylene-hydroxy.
In some embodiments according to any of the embodiments described herein, b is 3. In some embodiments, (i) P comprises at least one modified nucleoside X N; (ii) P comprises at least one modified internucleoside linker Y N, wherein at least one of T 1 or T 2 is S; or (iii) both (i) and (ii). In some embodiments, P comprises at least one dithiophosphate or phosphorothioate internucleoside linker. In some embodiments, P comprises 0, 1,2, or 3 phosphorodithioate internucleoside linkers. In some embodiments, P comprises a modified nucleoside selected from the group consisting of: 2' -O-alkyl nucleosides, 2' -O-alkoxyalkyl nucleosides, 2' -deoxynucleosides, and ribonucleosides. In some embodiments, the modified nucleoside is selected from the group consisting of: 5-bromo-2 '-O-methyluridine, 5-bromo-2' -deoxyuridine, 2 '-O-methyluridine, 2' -deoxyuridine, 2 '-O-methylthymidine, 2' -O-methylcytidine, 2'-O- (2-methoxyethyl) thymidine and 8-oxo-7, 8-dihydro-2' -deoxyguanosine. In some embodiments, X 5 ' is 5-bromo-2 ' -O-methyluridine, 5-bromo-2 ' -deoxyuridine, 2' -O-methyluridine, or 2' -deoxyuridine. In some embodiments, Y 3' or Y N at the 3' position of X 5' comprises an unsubstituted or substituted phosphorothioate. In some embodiments, Y PTE is
Wherein Z is O or S; d is an integer from 0 to 95; two on the right side of the structureIndicating the point of attachment to the adjacent nucleoside X N in the oligonucleotide P, and/>, to the left of the structureIndicating the point of connection with the joint L. In some embodiments, Y PTE is:
Wherein Z is O or S; d is an integer from 0 to 95; two on the right side of the structure Indicating the point of attachment to the adjacent nucleoside X N in the oligonucleotide P, and/>, to the left of the structureIndicating the point of connection with the joint L. In some embodiments, Z is S. In some embodiments, d is an integer from 1 to 25. In some embodiments, the linker L comprises a polyethylene glycol moiety. In some embodiments, the linker L is
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to Y PTE, and/>Indicating the point of attachment to the remainder of the conjugate. In some embodiments, P comprises one or more CpG sites. In some embodiments, the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the Q tag peptides are each linked to the C-terminus of one of the antibody heavy chains; and wherein one of the Q tag peptides is linked to an immunomodulatory oligonucleotide (P) via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide.
In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein the immunomodulatory oligonucleotide comprises the sequence of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein the immunomodulatory oligonucleotide comprises the sequence of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 87; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 87; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 87; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 87; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 75; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 87; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 89; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 88; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains comprises the amino acid sequence of SEQ ID No. 87; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; wherein each of the Q tag peptides (Q) comprises the amino acid sequence of SEQ ID No. 49; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 79; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein the immunomodulatory oligonucleotide comprises the sequence of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 79; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein the immunomodulatory oligonucleotide comprises the sequence of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In another aspect, provided herein is a conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 79; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein the immunomodulatory oligonucleotide comprises the sequence of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide. In some embodiments, the DAR of the conjugate is 1 or 2.
In another aspect, provided herein is a method for preparing a conjugate comprising (i) an antibody or antigen binding fragment thereof (Ab) that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), the method comprising: contacting the Ab with the oligonucleotide P in the presence of transglutaminase; wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47); wherein each P independently comprises the formula:
Wherein X 5' is a 5' terminal nucleoside; x 3' is a 3' terminal nucleoside; y PTE is an internucleoside phosphotriester; y 3' is a terminal phosphotriester; each X N is independently a nucleoside; each Y N is independently an internucleoside linker; b and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5; and L is a linker moiety comprising a terminal amine; and wherein the antibody or antigen binding fragment comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61). In another aspect, provided herein is a method for preparing a conjugate comprising (i) an antibody or antigen binding fragment thereof (Ab) that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47); wherein the antibody or antigen binding fragment comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to a Q-tag peptide via an amide bond to the glutamine residue of the Q-tag peptide and a linker (L), as shown in formula (A),
Wherein:
indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
each L is independently a bond or a linker moiety linked to Q via an amide bond with a glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide;
the method comprises contacting a compound of formula (B) with one or more immunomodulatory oligonucleotides P
Wherein Ab and Q are as defined above with respect to formula (a), and e is an integer from 1 to 20, wherein each P independently comprises the formula:
Wherein the method comprises the steps of
X 5 'is a 5' terminal nucleoside;
X 3 'is a 3' terminal nucleoside;
y PTE is an internucleoside phosphotriester;
y 3' is a terminal phosphotriester;
Each X N is independently a nucleoside;
each Y N is independently an internucleoside linker;
b and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5; and
L is a linker moiety comprising a terminal amine,
The contacting occurs in the presence of a transglutaminase. In some embodiments, each immunomodulation
The oligonucleotide is independently an oligonucleotide of formula (C) or formula (D) selected from the group consisting of the oligonucleotides of Table 15 and Table 17.
In some embodiments, each Q tag peptide sequence comprises peptide sequence RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Ab comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides (Q) having at least one glutamine residue; one Q tag peptide is attached to the C-terminus of each of the two antibody heavy chains. In some embodiments, the DAR of the conjugate is 1 or 2. In some embodiments, the DAR of the conjugate is 1, and the method further comprises isolating the conjugate with DAR 1 from the free oligonucleotide, unconjugated antibody, and the conjugate with DAR 2.
In another aspect, provided herein is a pharmaceutical composition comprising a conjugate of any of the above embodiments and a pharmaceutically acceptable carrier.
In another aspect, provided herein is a method of treating cancer, the method comprising administering to an individual an effective amount of a conjugate according to any of the above embodiments or a pharmaceutical composition according to any of the above embodiments. In another aspect, provided herein is a conjugate according to any of the above embodiments or a pharmaceutical composition according to any of the above embodiments in a method for treating cancer. In another aspect, provided herein is the use of a conjugate according to any of the above embodiments or a pharmaceutical composition according to any of the above embodiments in the manufacture of a medicament, for example for the treatment of cancer. In another aspect, provided herein is a method for activating bone marrow cells, the method comprising administering to an individual in need thereof an effective amount of a conjugate according to any of the above embodiments or a pharmaceutical composition according to any of the above embodiments. In another aspect, provided herein is a method for inducing TLR9 signaling in bone marrow cells, the method comprising administering to an individual in need thereof an effective amount of a conjugate according to any one of the above embodiments or a pharmaceutical composition according to any one of the above embodiments. In some embodiments, the individual has cancer. In some embodiments, the cancer is a liquid tumor. In other embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, squamous cell carcinoma, cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma), brain tumor, glioblastoma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, skin cancer, lung cancer, endometrial cancer, liver cancer, bladder cancer, gastric (or carcinoma of the stomach) (stomach cancer), pancreatic cancer, cervical cancer, ovarian cancer, urethral cancer, urothelial cancer, breast cancer, peritoneal cancer, uterine cancer, salivary gland cancer, renal cancer (kidney) or renal cancer (RENAL CANCER), prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer (testis) or testicular cancer (testicular cancer), melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin's lymphoma (non-Hodgkin's lymphoma; NHL), acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), mechl cell leukemia (MERKEL CELL), hairy cell leukemia, or chronic myelogenous leukemia (cmci). In some embodiments, the cancer is known or predicted to be unresponsive to PD-L1 or a PD-1 inhibitor (e.g., when administered as monotherapy, or when administered in the absence of an anti-SIRP-a antibody). In some embodiments, the individual does not achieve a significant therapeutic response to PD-L1 or a PD-1 inhibitor (e.g., an antibody that binds PD-L1 or PD-1). In some embodiments, the subject has been treated with PD-L1 or a PD-1 inhibitor (e.g., an antibody that binds PD-L1 or PD-1) prior to administration of the conjugate or composition. In some embodiments, the PD-L1 or PD-1 inhibitor is pembrolizumab, nivolumab, cimetidine Li Shan anti-rwlc, atrazumab, rituximab-gxly, dewaruzumab, or avistuzumab. In some embodiments, prior to administration of the conjugate or composition, the subject has been treated with PD-L1 or a PD-1 inhibitor (e.g., an antibody that binds PD-L1 or PD-1) and is not responsive to the treatment with the PD-L1 or PD-1 inhibitor (e.g., when administered as monotherapy, or when administered in the absence of an anti-SIRP-a antibody). In some embodiments, the cancer is, for example, SIRP- α expressing or overexpressing melanoma or renal cancer. In some embodiments, the cells of the cancer express human SIRP-a. In some embodiments, the cells of the cancer do not express human SIRP- α. In some embodiments, the method further comprises administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent comprises an immunotherapy, chemotherapy, radiation therapy, cell-based therapy, an anti-cancer vaccine, or an anti-cancer agent. In some embodiments, the methods further comprise administering to the individual PD-L1 or a PD-1 inhibitor (e.g., an antibody that binds PD-L1 or PD-1). In some embodiments, the method comprises administering to the individual PD-L1 or a PD-1 inhibitor, wherein: (a) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (b) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (c) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (d) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (e) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (f) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (g) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; (h) The antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide; or (i) the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein each of the antibody light chains comprises the amino acid sequence of SEQ ID No. 73; wherein each of the antibody heavy chains is linked to a C-terminal Q tag peptide (Q) and comprises the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide.
It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the detailed description below.
Drawings
The application may be understood by reference to the following description taken in conjunction with the accompanying drawings.
FIGS. 1A and 1B depict the activity of immunomodulatory oligonucleotides alone in human PBMC based on observed increases in expression of HLADR (FIG. 1A) and CD40 (FIG. 1B).
FIGS. 2A-2C show the effect of immunomodulatory oligonucleotides on increasing B cell numbers and activation. Figure 2A depicts the observed effect of various immunomodulatory polypeptides alone on B cell numbers. FIGS. 2B-2C depict the observed activation of B cells (via detection of CD40 expression) by immunomodulatory oligonucleotides alone.
Fig. 3 shows the percent yields of transglutaminase mediated binding and unbinding to polyethylene glycol linkers (-NH-C (=o) -PEG 23-NH2) and various Q tags. SEQ ID NOS 39-47 and 50-52 are shown.
FIGS. 4A and 4B show the percent change over time of the binding and unbinding of two Q-tag peptides (LSLSPGLLQGG, SEQ ID NO:39; and RPQGF, SEQ ID NO: 47) during transglutaminase binding.
FIG. 5 shows the activity of the indicated free CpG oligonucleotides on human PBMC as determined by CD40 expression on CD19+ B cells.
FIG. 6 shows the activity of the indicated free CpG oligonucleotides on human PBMC as determined by Ramos NFkb reporter gene assay.
Figures 7A-7C show the activity of the indicated free CpG oligonucleotides on human PBMCs from three different donor systems (D559, D804 and D643) as observed by CD40 expression.
FIG. 8 shows the activity of the indicated free CpG oligonucleotides on human PBMC as determined by CD40 expression on CD19+ B cells.
Fig. 9A-9D show schematic diagrams of exemplary conjugates, according to some embodiments. Exemplary antibodies with engineered Q-tags fused to the C-terminus of the heavy chain (RPQGFGPP; SEQ ID NO: 49) CpG conjugates are shown in FIG. 9A (DAR 1) and FIG. 9B (DAR 2). Exemplary antibodies with naturally occurring Q tags (Q295) exposed for binding via the N297A mutation are shown in fig. 9C (DAR 1) and fig. 9D (DAR 2).
FIGS. 10A-10D show activation of human bone marrow cells (e.g., monocytes and dendritic cells) by anti-SIRP-alpha antibody-CpG oligonucleotide conjugates. Activation was assessed by CD86 expression using flow cytometry.
FIGS. 11A-11D show stimulation of interferon regulatory factors (IRF 7; FIGS. 11A and 11B) and interleukin-6 (IL-6; FIGS. 11C and 11D) in human bone marrow cells (e.g., monocytes and dendritic cells) by anti-SIRP-alpha antibody-CpG oligonucleotide conjugates.
FIGS. 12A-12D show stimulation of cytokine secretion in human PBMC by anti-SIRP-alpha antibody-CpG oligonucleotide conjugates. Secretion of IFN-. Alpha.2 (FIG. 12A), IFN-. Gamma. (FIG. 12B), IL-6 (FIG. 12C) and IL-10 (FIG. 12D) is shown.
FIG. 13 shows the ability of CpG oligonucleotides to activate CD40 expression in bone marrow cells. The effect on cd14+ monocytes (left) and dendritic cells (right) is shown.
FIG. 14 shows stimulation of tumor phagocytosis by monocyte-derived M2 macrophages after treatment with anti-SIRP-alpha antibody-CpG oligonucleotide conjugates compared to unconjugated antibody or media controls.
FIGS. 15A and 15B show the anti-tumor effect in RENCA (SIRP-alpha positive) isogenic tumor models using anti-SIRP-alpha antibody-CpG oligonucleotide conjugates of either mouse IgG2a or mouse IgG1 Fc regions.
Figures 16A and 16B show activation of monocytes when PBMCs were co-cultured by three anti-SIRP-a antibody-CpG oligonucleotide conjugates each conjugated to a different Fc domain (human IgG4, human IgG1 and human IgG 1-AAA) in the presence of a SIRP-a positive or SIRP-a negative tumor cell line, as compared to unconjugated antibodies. The DLD-1 cells in FIG. 16A were transduced to overexpress SIRP-alpha (SIRP-alpha positive), whereas the parent DLD-1 cells in FIG. 16B did not express SIRP-alpha (SIRP-alpha negative).
FIG. 17A shows a diagram of an anti-SIRP-alpha antibody-CpG oligonucleotide conjugate.
Figures 17B-17D show that anti-SIRP-a antibody-CpG oligonucleotide conjugates are potent TLR9 agonists with cross-species targeted activation. Human PBMC (fig. 17B), cynomolgus PBMC (fig. 17C) or mouse splenocytes (fig. 17D) were stimulated with anti-hsrp-a antibodies, anti-hsrp-a antibody-CpG oligonucleotide conjugates, cpG 7-7 oligonucleotides or anti-mSIRP-a antibody-CpG oligonucleotide conjugates with murine-reactive mT-CpG for 24 hours or 48 hours and surface marker expression was determined by flow cytometry.
Figures 18A and 18B show that anti-SIRP-a antibody-CpG oligonucleotide conjugates specifically target and activate SIRP-a positive immune cells in human PBMC cultures. Human PBMCs were stimulated with anti-SIRP-a antibody-CpG oligonucleotide conjugate or anti-CD 22 antibody-CpG oligonucleotide conjugate for 24 hours and surface marker expression on monocytes (fig. 18A) and B cells (fig. 18B) was determined by flow cytometry.
FIGS. 19 and 20 show activation of bone marrow cells co-cultured with SIRP-alpha positive DLD-1 tumor cells (FIG. 19) versus SIRP-alpha negative DLD-1 tumor cells (FIG. 20).
FIGS. 21A and 21B show phagocytosis of SIRP-alpha positive DLD-1 tumor cells (FIG. 21A) or SIRP-alpha negative DLD-1 tumor cells (FIG. 21B) in the presence of human monocyte-derived macrophages and an anti-SIRP-alpha antibody-CpG oligonucleotide conjugate or an anti-SIRP-alpha antibody. Phagocytosis% was determined by flow cytometry.
FIGS. 22A and 22B show the anti-tumor activity of anti-SIRP-alpha antibody-CpG oligonucleotide conjugates in a mouse isogenic model. Mice carrying SIRP- α -expressing MC38 (fig. 22A) or parental MC38 cells (fig. 22B) were dosed intraperitoneally (i.p.) twice, three days apart from 1mg/kg of anti-mSIRP- α antibody conjugated to murine reactive mT-CpG (square) or PBS control (triangle). Arrows indicate the doses administered.
FIGS. 23A and 23B show single dose anti-tumor activity of anti-SIRP-alpha antibody-CpG oligonucleotide conjugates in a mouse RENCA model. In fig. 23A, mice bearing RENCA tumor cells were dosed intraperitoneally (i.p.) three times, three days apart from 10mg/kg of anti-mSIRP-a antibody (square) or PBS (triangle) that bound murine reactive mT-CpG. In FIG. 23B, individual groups were dosed three times with 10mg/kg of anti-PD-1 (triangle) three days apart or PBS (circle). Arrows indicate the doses administered.
FIG. 24A shows the effect of anti-SIRP-alpha antibody-CpG oligonucleotide conjugates in a mouse CT26 isogenic tumor model. Mice bearing CT26 tumor cells were treated intraperitoneally (i.p.) with 1mg/kg of anti-mSIRP-alpha antibody conjugated to murine reactive mT-CpG or PBS control. Arrows indicate the doses administered.
Fig. 24B shows the results of subsequent studies in a mouse CT26 syngeneic tumor model. Mice bearing CT26 tumor cells were treated with a suboptimal dose of 0.3mg/kg of anti-SIRP-a antibody-CpG oligonucleotide conjugate, 10mg/kg of anti-PD-1 antibody, a combination of both, or PBS control (fig. 24B). The anti-SIRP-alpha antibody-CpG oligonucleotide conjugate has a heavy chain comprising the sequence of SEQ ID NO:91 and a light chain comprising the sequence of SEQ ID NO:111, conjugated to a mouse CpG oligonucleotide 4523 (SEQ ID NO: 121). Arrows indicate the doses administered. The unpaired t-test was used to calculate the P-value of the comparative combination group versus anti-PD-1.
FIG. 25 shows the anti-tumor activity of a combination of an anti-SIRP-alpha antibody conjugate and an anti-PD-L1 antibody in a mouse syngeneic tumor model. Mice bearing B16F10 tumor cells were treated intraperitoneally (i.p.) with 30mg/kg of anti-SIRP-a antibody conjugated to murine reactive mT-CpG4523, 10mg/kg of anti-PD-L1 antibody, a combination of both, or PBS control.
FIG. 26 shows the anti-tumor activity of anti-SIRP-alpha antibody conjugates in mice that were non-responsive to previous anti-PD-1 treatments. Mice bearing CT26 colon cancer cells were treated with PBS or anti-PD-1 (10 mg/kg). If the tumor measurement exceeds the initial size and is greater than 250mm 3, the anti-PD-1 treated mice are considered non-responders. Mice with an average tumor volume size of 338mm 3 on day 11 that were unresponsive to anti-PD-1 were again randomized into 3 new treatment groups: 1mg/kg of anti-SIRP-alpha antibody conjugate alone (group 1); a combination of 1mg/kg anti-SIRP-a antibody conjugate with 10mg/kg anti-PD-1 (group 2); or 10mg/kg anti-PD-1 monotherapy (group 3). Treatments were administered intraperitoneally every 3 days at a total of 2 doses. Mean tumor volume (mm 3) ±sem over time was plotted for each group. Mpk = mg/kg.
FIGS. 27A and 27B show activation of TLR9 pathway signaling by an anti-SIRP-alpha antibody or an anti-SIRP-alpha antibody, cpG oligonucleotide conjugate, as compared to CpG oligonucleotide alone. TLR9 activation was also assessed in 2 ways: by monitoring the Interferon Regulatory Factor (IRF) pathway that induces the activity of secreting luciferin (fig. 27A), and by monitoring the NF-kB pathway that induces the activity of Secreting Embryonic Alkaline Phosphatase (SEAP) (fig. 27B). Results are expressed as fold induction versus concentration relative to the medium control.
Detailed Description
The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present invention, but is instead provided as a description of exemplary embodiments.
As discussed above, cpG ODNs are generally prone to degradation in serum and/or exhibit non-uniform tissue distribution, off-target activity, and localized toxicity in vivo; thus, its pharmacokinetic properties may limit its development as a potential therapeutic agent.
One solution is to conjugate an immunomodulatory oligonucleotide (e.g., cpG ODN) with a targeting moiety that specifically targets a tissue or cell to overcome the uneven distribution of the oligonucleotide. See US2018/0312536. In particular, transglutaminase mediated reactions can be used to conjugate polypeptide targeting moieties containing glutamine residues with CpG ODNs containing primary amine groups. Microbial transglutaminase (mTG) is derived from streptomyces mobaraensis (Streptomyces mobaraensis). mTG catalyzes the transamidation reaction between the 'reactive' glutamine and the 'reactive' lysine residues of the protein under pH-controlled aqueous conditions (including physiological conditions), but the 'reactive' lysine residues can also be simple low molecular weight primary amines such as 5-aminopentyl. For endogenous glutamine on a protein to be recognized as a mTG substrate, two criteria appear to be important: 1) The presence of hydrophobic amino acids adjacent to glutamine residues in the peptide sequence, and 2) the localization of glutamine to the loop, wherein local chain flexibility enhances reactivity to mTG.
The present invention is based at least in part on the following findings: the anti-SIRP-alpha antibody-CpG oligonucleotide conjugates are effective in activating human bone marrow cells (e.g., monocytes and dendritic cells), stimulating cytokine production, and stimulating tumor cell phagocytosis. The manner in which these conjugates are prepared is also described. In particular, conjugation may be performed by a Transglutaminase (TG) -mediated reaction. Intermediate compounds useful in the preparation of these conjugates are also provided.
The present disclosure demonstrates that anti-SIRP-a antibody-CpG oligonucleotide conjugates promote bone marrow cell (e.g., monocytes and dendritic cells) activation, activate both IRF7 and NFkB pathways, stimulate cytokine production (e.g., IL-6, IFN- α2, IFN- γ, and IL-10), and induce tumor cell phagocytosis by monocyte-derived macrophages. In addition, anti-SIRP-alpha antibody-CpG oligonucleotide conjugates were found to mediate anti-tumor activity. Such conjugates were found to integrate TLR9 activation and blocking of CD 47-SIRP-a interaction with bone marrow cells such that the anti-tumor immune response was from both the innate and adaptive immune systems. anti-SIRP-alpha antibody-CpG oligonucleotide conjugates have the additional benefit of preferential tumor cell targeting in SIRP-alpha expression (SIRP-alpha positive) tumor models.
I. Definition of the definition
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications mentioned herein are incorporated by reference in their entirety. If the definition set forth in this section is contrary to or otherwise inconsistent with the definition set forth in the patent, application, or other publication, which is incorporated by reference, then the definition set forth in this section prevails over the definition that is incorporated by reference.
It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. This disclosure specifically covers and discloses herein all combinations of embodiments relating to particular method steps, reagents or conditions, as if each combination were individually and specifically disclosed.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for recitation of claim elements using exclusive terminology such as "only (solely)", "only (only)", and the like, or using "negative" limitation.
Throughout this application, reference to compounds of formulas (a) - (D) includes ionic, polymorphic, pseudopolymorphic, amorphous, solvate, co-crystal, chelate, isomer, tautomer, oxide (e.g., N-oxide, S-oxide), ester, prodrug, isotope, and/or protected forms thereof unless the context indicates otherwise. In some embodiments, reference to compounds of formulas (a) - (D) includes polymorphs, solvates, co-crystals, isomers, tautomers, and/or oxides thereof. In some embodiments, mention of compounds of formulas (a) - (D) includes polymorphs, solvates, and/or co-crystals thereof. In some embodiments, mention of compounds of formulas (a) - (D) includes isomers, tautomers, and/or oxides thereof. In some embodiments, mention of compounds of formulas (a) - (D) includes solvates thereof.
"Alkyl" encompasses straight and branched carbon chains having the indicated number of carbon atoms, for example 1 to 20 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms. For example, C 1-6 alkyl encompasses straight and branched alkyl groups of 1 to 6 carbon atoms. When naming an alkyl residue having a specific number of carbons, all branched and straight chain forms having said number of carbons are intended to be covered; thus, for example, "propyl" includes n-propyl and isopropyl; and "butyl" includes n-butyl, sec-butyl, isobutyl and tert-butyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl.
When a range of values (e.g., C 1-6 alkyl) is given, each value within the range as well as all intervening ranges are encompassed. For example, "C 1-6 alkyl" includes C1、C2、C3、C4、C5、C6、C1-6、C2-6、C3-6、C4-6、C5-6、C1-5、C2-5、C3-5、C4-5、C1-4、C2-4、C3-4、C1-3、C2-3 and C 1-2 alkyl.
"Alkenyl" refers to an unsaturated branched or straight chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one carbon-carbon double bond. The groups may be in cis or trans configuration (Z or E configuration) around the double bond. Alkenyl groups include, but are not limited to, ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl) and butenyl (e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl).
"Alkynyl" refers to an unsaturated branched or straight chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one carbon-carbon triple bond. Alkynyl groups include, but are not limited to, ethynyl, propynyl (e.g., prop-1-yn-1-yl, prop-2-yn-1-yl) and butynyl (e.g., but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl).
As used herein, the term "amino" means-N (R N1)2 wherein each of the substituents may itself be unsubstituted or substituted with an unsubstituted substituent for each respective group as defined herein, or if the amino group is substituted, each R N1 is independently H、-OH、-NO2、-N(RN2)2、-SO2ORN2、-SO2RN2、-SORN2、-COORN2、N- protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl, cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least one R N1 is not H, and wherein each R N2 is independently H, alkyl, or aryl.
The term "immunomodulatory oligonucleotide" as used herein refers to an oligonucleotide construct containing a total of 6 to 50 consecutive nucleosides covalently bound together by internucleoside bridging groups independently selected from the group consisting of internucleoside phosphates and optionally internucleoside abasic spacers. The immunomodulatory oligonucleotide is capped at the 5 'end and 3' end with 5 'and 3' end capping groups, respectively. Immunomodulatory oligonucleotides are capable of modulating an innate immune response as determined by: for example, in an immune cell (e.g., an antigen presenting cell) to which the immunomodulatory oligonucleotide is delivered (as compared to another immune cell (e.g., an antigen presenting cell) to which the immunomodulatory oligonucleotide is not delivered), or in an immune cell that interacts (including direct intercellular interactions, as well as direct stimulation of one or more cytokines secreted by, for example, the cell to which the immunomodulatory oligonucleotide is delivered), a change in activation of an intracellular signaling pathway (including, but not limited to, nfkb), a change in expression of an activation marker, or a change in secretion of at least one inflammatory cytokine or at least one type I interferon. The immunomodulatory oligonucleotide may contain a conjugate group or, if the immunomodulatory oligonucleotide is part of a conjugate, a linker that is bound to a targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary moieties (e.g., polyethylene glycol). The conjugate group or linker may be part of a phosphotriester or a terminal end-capping group.
The term "immunostimulatory oligonucleotide" as used herein refers to an immunomodulatory oligonucleotide capable of activating an immune response, as determined by: in immune cells (e.g., antigen presenting cells) to which the immunostimulatory oligonucleotide is delivered (e.g., as compared to another immune cell (e.g., antigen presenting cells) to which the immunostimulatory oligonucleotide is not delivered), or in immune cells that interact (including direct intercellular interactions, as well as direct stimulation of, for example, one or more cytokines secreted by the cell to which the immunostimulatory oligonucleotide is delivered), an increase in activation of an intracellular signaling pathway (such as nfkb), or an increase in the level of an activated or functional cell surface marker, or an increase in the secretion of at least one inflammatory cytokine or at least one type I interferon. In some embodiments, the immunostimulatory oligonucleotide contains at least one cytidine-p-guanosine (CpG) sequence, where p is an internucleoside phosphodiester (e.g., a phosphate or phosphorothioate) or an internucleoside phosphotriester or phosphorothioate triester. As used herein, cpG-containing immunostimulatory oligonucleotides may be naturally occurring, such as CpG ODNs of bacterial or viral origin, or synthetic. For example, in some embodiments, the CpG sequences in the immunostimulatory oligonucleotide contain 2' -deoxyribose.
The term "immunosuppressive oligonucleotide" as used herein refers to an immunomodulatory oligonucleotide capable of antagonizing an immune response, as determined by: in immune cells (e.g., antigen presenting cells) to which the immunostimulatory oligonucleotide is delivered (e.g., as compared to another immune cell (e.g., antigen presenting cells) to which the immunostimulatory oligonucleotide is not delivered), or in immune cells that interact (including direct intercellular interactions, as well as direct stimulation of one or more cytokines secreted, for example, by cells to which the immunostimulatory oligonucleotide is delivered) with immune cells (e.g., antigen presenting cells) to which the immunostimulatory oligonucleotide is delivered, reduced or absent activation of nfkb, or increased levels of functionally activated cell surface markers, or increased or absent secretion of at least one inflammatory cytokine or at least one type I interferon.
It should be understood that the terms "oligonucleotide" and "polynucleotide" are used interchangeably herein. It is further understood that the terms "immunomodulatory oligonucleotide", "immunostimulatory oligonucleotide", "immunosuppressive oligonucleotide" and "conjugate" encompass salts of immunomodulatory oligonucleotides, immunostimulatory oligonucleotides, immunosuppressive oligonucleotides and conjugates, respectively. For example, the terms "immunomodulatory oligonucleotide", "immunostimulatory oligonucleotide", "immunosuppressive oligonucleotide" and "conjugate" encompass both phosphate, phosphorothioate or phosphorodithioate in protonated neutral form (P-XH moiety wherein X is O or S) and phosphate, phosphorothioate or phosphorodithioate in deprotonated ionic form (P-X - moiety wherein X is O or S). Thus, it is understood that phosphate and phosphodiester described as having one or more of R E1、RE2 and R E3 as hydrogen encompass salts in which the phosphate, phosphorothioate or phosphorodithioate is present in the deprotonated ionic form. In addition, referring to immunomodulatory oligonucleotides, immunostimulatory oligonucleotides, immunosuppressive oligonucleotides, and/or oligonucleotides (e.g., cpG oligonucleotides), the terms "free", "bare" and "unconjugated" may be used interchangeably herein.
As used herein, the term "phosphotriester" refers to a phosphate ester in which all three valences are substituted with non-hydrogen substituents. The phosphotriester consists of: phosphate, phosphorothioate or phosphorodithioate; one or two linkages to nucleoside or abasic spacers and/or phosphoryl groups; and one or two groups independently selected from the group consisting of: a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; and a linker bonded to the targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary moieties. The terminal phosphotriester includes one bond to a nucleoside-containing group and two groups independently selected from the group consisting of: a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a phosphoryl group; and a linker bonded to the targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary moieties. In some embodiments, the terminal phosphotriester contains 1 or 0 linkers that are bound to the targeting moiety and optionally to one or more (e.g., 1 to 6) auxiliary moieties. The internucleoside phosphate comprises two linkages to nucleoside-containing groups. The phosphotriester may be a group of the structure:
/>
wherein:
Each of X E1 and X E2 is independently O or S;
Each R E1 and R E3 is independently a bond to a nucleoside; sugar analogs without base spacers; a bioreversible group; a non-bioreversible group; an auxiliary portion; a binding group; a linker bonded to the targeting moiety; a linker bonded to the targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; or a phosphorus atom in a group of the formula-P (=X E1)(-XE2-RE2 A) -O-,
Wherein R E2 a is hydrogen; a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; or a linker bonded to the targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties; and
R E2 is a bioreversible group; a non-bioreversible group; an auxiliary portion; a conjugate group; a linker bonded to the targeting moiety; or a linker bonded to the targeting moiety and one or more (e.g., 1 to 6) auxiliary moieties;
Provided that at least one of R E1 and R E3 is a bond to a group containing at least one nucleoside.
If both R E1 and R E3 are bonds to groups containing at least one nucleoside, the phosphotriester is an internucleoside phosphotriester. If one and only one of R E1 and R E3 is a bond to a nucleoside containing group, the phosphotriester is a terminal phosphotriester.
As used herein, the term "amino acid" refers to any amino acid (both standard and non-standard amino acids) including, but not limited to, alpha-amino acids, beta-amino acids, gamma-amino acids, and delta-amino acids. Examples of suitable amino acids include, but are not limited to, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. Additional examples of suitable amino acids include, but are not limited to, ornithine, hypusine (hypusine), 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid, citrulline, beta-alanine, alpha-ethyl-glycine, alpha-propyl-glycine, and norleucine.
The terms "antibody," "immunoglobulin," and "Ig" are used interchangeably herein and are used in the broadest sense and specifically cover, for example, individual monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions having multi-or mono-epitope specificity, polyclonal or monoclonal antibodies, multivalent antibodies, multi-specific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain antibodies, and antibody fragments. Antibodies can be human, humanized, chimeric and/or affinity matured antibodies, as well as antibodies from other species (e.g., mice and rabbits).
The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides capable of binding to a particular antigen and consisting of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), and each amino-terminal portion of each chain comprises a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain comprises a constant region. See Borrebaeck (ed) (1995) Antibody Engineering second edition, oxford University press; kuby (1997) third edition of Immunology, w.h. freeman and Company, new York. Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments thereof, which refer to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments of antibodies include single chain Fv (scFv) (e.g., including monospecific or bispecific), fab fragments, F (ab ') fragments, F (ab) 2 fragments, F (ab') 2 fragments, disulfide-linked Fv (sdFv), fd fragments, fv fragments, scRv-Fc, nanobodies, diabodies, triabodies, tetrafunctional antibodies, and minibodies. In some embodiments, the antibody comprises an Fc variant with reduced or eliminated effector function. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen binding domains or molecules (e.g., one or more Complementarity Determining Regions (CDRs) of an anti-CD 56 antibody or an anti-sirpa antibody) that contain an antigen binding site that binds an antigen. Such antibody fragments are described, for example, in the following: harlow and Lane, antibodies A Laboratory Manual, cold Spring Harbor Laboratory, new York (1989); myers (ed.), molecular and Biotechnology:A Comprehensive DESK REFERENCE, new York: VCH Publisher, inc.; huston et al, cell Biophysics 1993,22,189-224; pluckthun and Skerra, meth. Enzymol.1989,178,497-515; and Day, advanced Immunochemistry, second edition, wiley-lists, inc., new York, NY (1990). Antibodies provided herein can be of any type (e.g., igG, igE, igM, igD, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or any subclass (e.g., igG2a and IgG2 b) of immunoglobulin molecules.
The term "antigen" refers to a predetermined target to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or fragment thereof, or other naturally occurring or synthetic compound. In one embodiment, the target antigen is a polypeptide.
The terms "antigen binding fragment," "antigen binding domain," and "antigen binding region" refer to a portion of an antibody that comprises amino acid residues that interact with an antigen (e.g., a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or fragment thereof, or other naturally occurring or synthetic compound) and confer specificity and affinity to the binding agent for the antigen (e.g., a Complementarity Determining Region (CDR)).
The terms "specifically binds", "specifically binds to" or "is specific for" a particular polypeptide or an epitope on a particular polypeptide target may be revealed, for example, by a molecule (e.g., an antibody) having a dissociation constant (K d) for the target of at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M, at least about 10 - 11 M, or at least about 10 -12 M. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
The 4-chain antibody unit is an iso-tetralin protein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) of each alpha and gamma chain and four CH domains of the mu and epsilon isoforms. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH and CL is aligned with the first constant domain of the heavy chain (CH 1). It is believed that a particular amino acid residue forms an interface between the light chain variable domain and the heavy chain variable domain. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., basic AND CLINICAL Immunology, 8 th edition, stites et al (ed.), appleton & Lange, norwalk, CT,1994, page 71 and chapter 6.
The term "variable region" or "variable domain" refers to a portion of the light or heavy chain of an antibody that is typically located at the amino terminus of the light or heavy chain and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region differ widely in terms of the sequence in an antibody. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is unevenly distributed over the 110 amino acid span of the variable region. In practice, the V region consists of: a weakly variable (e.g., relatively constant) stretch of about 15-30 amino acids (referred to as a Framework Region (FR)) separated by a shorter, more variable (e.g., extremely variable) region (referred to as a "hypervariable region", each about 9-12 amino acids in length). The variable regions of the heavy and light chains each comprise four FR joined by three hypervariable regions, which generally adopt a β -sheet configuration, the hypervariable regions forming loops connecting the β -sheet structure and in some cases forming part of the β -sheet structure. The hypervariable regions in each chain are tightly held together by the FR and together with the hypervariable region from the other chain contribute to the formation of the antigen binding site of the antibody (see, e.g., kabat et al Sequences of Proteins of Immunological Interest, 5 th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary widely in sequence between different antibodies. The variability in the sequence is concentrated in the CDRs, while the less variable part of the variable region is called the Framework Region (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of antibodies with antigens. In a particular embodiment, the variable region is a human variable region.
The term "variable region residue number as in Kabat" or "amino acid position number as in Kabat" and variants thereof refer to the numbering system of the heavy chain variable region or the light chain variable region used in antibody compilation in Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991). Using such numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening of, or insertion into, the FR or CDR of the variable domain. For example, the heavy chain variable domain can include a single amino acid insertion (according to Kabat, residue 52 a) following residue 52 of H2 and an insertion residue (e.g., according to Kabat, residues 82a, 82b, and 82c, etc.) following heavy chain FR residue 82. For a given antibody, the Kabat numbering of residues may be determined by aligning the homologous regions of the antibody sequences with a "standard" Kabat numbering sequence. The Kabat numbering system is generally used in reference to residues in the variable domain (residues 1-107 of the general light chain and residues 1-113 of the heavy chain) (e.g., kabat et al, sequences of Immunological Interest. 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in the same document as Kabat et al). "EU index as in Kabat" refers to the residue numbering of the human IgG 1EU antibody. Other numbering systems have been described, including, for example AbM, chothia, contact, IMGT and AHon.
An "intact" antibody is an antibody comprising an antigen binding site, CL and at least heavy chain constant regions CH1, CH2 and CH 3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
The term "antibody fragment" refers to a portion of an intact antibody, preferably the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments; diabodies and diabodies (see, e.g., holliger et al, proc. Natl. Acad. Sci. U.S. A.1993,90,6444-8; lu et al, J. Biol. Chem.2005,280,19665-72; hudson et al, nat. Med.2003,9,129-134; WO 93/11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single chain antibody molecules (see, e.g., U.S. Pat. nos. 4,946,778, 5,260,203, 5,482,858, and 5,476,786); a double variable domain antibody (see, e.g., U.S. patent 7,612,181); single variable domain antibodies (SdAb) (see, e.g., woolven et al, immunogenetics1999,50,98-101; streltsov et al, proc.Natl. Acad. Sci. U.S. A.2004,101, 12444-12449); and multispecific antibodies formed from antibody fragments.
The term "functional fragment", "binding fragment" or "antigen-binding fragment" of an antibody refers to a molecule that exhibits at least one biological function elicited by an intact antibody, said function comprising at least binding to a target antigen.
When used in reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion including a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isoforms), referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains vary in size: alpha, delta and gamma contain approximately 450 amino acids, while mu and epsilon contain approximately 550 amino acids. These different types of heavy chains, when combined with light chains, produce five well-known classes (e.g., isotypes) of antibodies, igA, igD, igE, igG and IgM, respectively, including the four subclasses of IgG, namely IgG1, igG2, igG3, and IgG4. The heavy chain may be a human heavy chain.
When used in reference to an antibody, the term "light chain" refers to a polypeptide chain of about 25kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (kappa) or lambda (lambda). The light chain amino acid sequences are well known in the art. The light chain may be a human light chain.
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 naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on an antigen. In particular embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell, wherein the antibody binds only to a βcloxol epitope (beta klotho epitope), e.g., as determined by ELISA or other antigen binding or competitive binding assays known in the art. The term "monoclonal" is not limited to any particular method for producing antibodies. For example, monoclonal antibodies suitable for use in the present disclosure may be prepared by the hybridoma method described for the first time by Kohler et al, nature 1975,256,495, or may be prepared using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, nature1991,352,624-628 and Marks et al, J.mol. Biol.1991,222, 581-597. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, e.g., short Protocols in Molecular Biology, (2002) 5 th edition, ausubel et al, john Wiley and Sons, chapter 11 in New York). Exemplary methods of producing monoclonal antibodies are provided in the examples herein.
A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that includes a human immunoglobulin (e.g., recipient antibody) in which the natural CDR residues are replaced by residues from the corresponding CDRs of a non-human species (e.g., donor antibody), such as mouse, rat, rabbit, or a non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further improve antibody efficacy. The humanized antibody heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin and all or substantially all of the FR are FR of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al, nature 1986,321,522-525; riechmann et al, nature 1988,332,323-329; presta, curr. Opin. Biotechnol.1992,3,394-398; carter et al, proc.Natl.Acad.Sci.U.S. A.1992,89,4285-4289; and U.S. Pat. nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213 and 6,054,297.
A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human and/or has been prepared using any of the human antibody preparation techniques as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol.1991,227,381; marks et al, J.Mol. Biol.1991,222, 581) and yeast display libraries (Chao et al, nature Protocols2006,1, 755-768). The methods described below are also useful methods for preparing human monoclonal antibodies: cole et al Monoclonal Antibodies AND CANCER THERAPY, alan R.Lists, page 77 (1985); boerner et al, J.Immunol.1991,147,86-95. See also van Dijk and VAN DE WINKEL, curr. Opin. Pharmacol.2001,5,368-374. Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., a mouse) that has been modified to produce such antibodies in response to antigen stimulation, but whose endogenous locus has been disabled (see, e.g., jakobovits, curr. Opin. Biotechnol.1995,6,561-566; bru ggemann and Taussing, curr. Opin. Biotechnol.1997,8,455-458; and U.S. Pat. Nos. 6,075,181 and 6,150,584, regarding XENOMOUSE TM techniques). See also, e.g., li et al, proc.Natl.Acad.Sci.U.S.A.2006,103,3557-3562 for human antibodies produced via human B cell hybridoma technology.
"CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework regions of an immunoglobulin (Ig or antibody) VH β -sheet framework, or one of the three hypervariable regions (L1, L2 or L3) within the non-framework regions of an antibody VL β -sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, kabat as the region of highest variability within the variable (V) domains of antibodies. Kabat et al, J.biol. Chem.1977,252,6609-6616; kabat, adv.protein chem.1978,32,1-75.CDR region sequences are also defined structurally by Chothia as those residues that are not part of the conserved β -sheet framework and are therefore able to accommodate different conformations. Chothia and Lesk, J.mol.biol.1987,196,901-917. Both terms are recognized in the art. CDR region sequences are also defined by AbM, contact and IMGT. CDR positions within the variable regions of typical antibodies have been determined by comparing a number of structures. Al-Lazikani et Al, J.mol. Biol.1997,273,927-948; morea et al, methods 2000,20,267-279. Since the number of residues within a hypervariable region varies from antibody to antibody, other residues relative to typical positions are conventionally numbered with the residue numbers in the numbering scheme of a, b, c, etc., next to the typical variable region. Al-Lazikani et Al, supra (1997). Such nomenclature is also well known to those skilled in the art.
In this context, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy-terminus. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may be removed, for example during preparation or purification of the antibody, or by engineering the nucleic acid encoding the heavy chain of the antibody in a recombinant manner. Thus, a composition of intact antibodies may comprise a population of antibodies with all K447 residues removed, a population of antibodies with no K447 residues removed, and a population of antibodies with a mixture of antibodies with and without K447 residues.
"Cycloalkyl" refers to a non-aromatic fully saturated carbocyclic ring having the indicated number of carbon atoms (e.g., 3 to 10, or 3 to 8, or 3 to 6 ring carbon atoms). Cycloalkyl groups may be monocyclic or polycyclic (e.g., bicyclic, tricyclic). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, as well as bridged and caged ring groups (e.g., norbornane, bicyclo [2.2.2] octane). In addition, one ring of the polycyclic cycloalkyl group may be aromatic, provided that the polycyclic cycloalkyl group is bonded to the parent structure via a non-aromatic carbon. For example, 1,2,3, 4-tetrahydronaphthalen-1-yl (wherein the moiety is bonded to the parent structure via a non-aromatic carbon atom) is cycloalkyl, while 1,2,3, 4-tetrahydronaphthalen-5-yl (wherein the moiety is bonded to the parent structure via an aromatic carbon atom) is not considered cycloalkyl. Examples of polycyclic cycloalkyl groups consisting of cycloalkyl groups fused to aromatic rings are described below.
"Cycloalkenyl" refers to a non-aromatic carbocyclic ring containing the indicated number of carbon atoms (e.g., 3 to 10, or 3 to 8, or 3 to 6 ring carbon atoms) and at least one carbon-carbon double bond. Cycloalkenyl groups can be monocyclic or polycyclic (e.g., bicyclic, tricyclic). Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl and cyclohexenyl, as well as bridged and caged ring groups (e.g., bicyclo [2.2.2] octene). In addition, one ring of the polycycloalkenyl group may be aromatic, provided that the polycycloalkenyl group is bonded to the parent structure via a non-aromatic carbon atom. For example, inden-1-yl (where the moiety is bonded to the parent structure via a non-aromatic carbon atom) is considered a cycloalkenyl group, while inden-4-yl (where the moiety is bonded to the parent structure via an aromatic carbon atom) is not considered a cycloalkenyl group. Examples of polycyclic cycloalkenyl groups consisting of cycloalkenyl groups fused to aromatic rings are described below.
"Cycloalkynyl" refers to an unsaturated hydrocarbon radical within a cycloalkyl having at least one site of alkyne unsaturation (i.e., having at least one moiety of formula c≡c). A cycloalkynyl group may consist of one ring (such as cyclooctyne) or multiple rings. One cycloalkynyl moiety is an unsaturated cyclic hydrocarbon having 5 to 10 ring carbon atoms ("C 5-C10 cycloalkynyl"). Examples include cyclopentyne, cyclohexenyne, cycloheptyne, cyclooctyne, cyclononene, and the like.
"Aryl" refers to an aromatic carbocyclic ring having the indicated number of carbon atoms (e.g., 6 to 12 or 6 to 10 carbon atoms). Aryl groups may be monocyclic or polycyclic (e.g., bicyclic, tricyclic). In some cases, both rings of the polycyclic aryl group are aromatic (e.g., naphthyl). In other cases, the polycyclic aryl group may include a non-aromatic ring fused to an aromatic ring, provided that the polycyclic aryl group is bonded to the parent structure via an atom in the aromatic ring. Thus, 1,2,3, 4-tetrahydronaphthalen-5-yl (wherein the moiety is bonded to the parent structure via an aromatic carbon atom) is considered an aryl group, whereas 1,2,3, 4-tetrahydronaphthalen-1-yl (wherein the moiety is bonded to the parent structure via a non-aromatic carbon atom) is not considered an aryl group. Similarly, 1,2,3, 4-tetrahydroquinolin-8-yl (wherein the moiety is bonded to the parent structure via an aromatic carbon atom) is considered an aryl group, while 1,2,3, 4-tetrahydroquinolin-1-yl (wherein the moiety is bonded to the parent structure via a non-aromatic nitrogen atom) is not considered an aryl group. However, as defined herein, the term "aryl" does not encompass or overlap with "heteroaryl" regardless of the point of attachment (e.g., quinolin-5-yl and quinolin-2-yl are both heteroaryl). In some cases, aryl is phenyl or naphthyl. In some cases, aryl is phenyl. Additional examples of aryl groups comprising aromatic carbocycles fused to non-aromatic rings are described below.
The term "DAR" refers to the drug-antibody ratio of an oligonucleotide-antibody conjugate, more particularly the immunomodulatory oligonucleotide-antibody ratio. In some cases, for example, an oligonucleotide-antibody conjugate may be described herein as having a DAR of 1 or as a DAR1 conjugate, wherein the oligonucleotide-antibody ratio is 1:1. In other cases, an oligonucleotide-antibody conjugate may be described herein as having a DAR of 2 or as a DAR2 conjugate, wherein the oligonucleotide-antibody ratio is 2:1.
"Heteroaryl" means an aromatic ring containing the indicated number of atoms (e.g., 5-to 12-membered or 5-to 10-membered heteroaryl) consisting of one or more heteroatoms (e.g., 1,2, 3, or 4 heteroatoms) selected from N, O and S, with the remaining ring atoms being carbon. Heteroaryl groups do not contain adjacent S and O atoms. In some embodiments, the total number of S and O atoms in the heteroaryl group is no more than 2. In some embodiments, the total number of S and O atoms in the heteroaryl group does not exceed 1. Unless otherwise indicated, heteroaryl groups may be bonded to the parent structure through a carbon or nitrogen atom, where valency permits. For example, "pyridyl" includes 2-pyridyl, 3-pyridyl and 4-pyridyl, and "pyrrolyl" includes 1-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl.
In some cases, the heteroaryl group is monocyclic. Examples include pyrrole, pyrazole, imidazole, triazole (e.g., 1,2, 3-triazole, 1,2, 4-triazole), tetrazole, furan, isoxazole, oxazole, oxadiazole (e.g., 1,2, 3-oxadiazole, 1,2, 4-oxadiazole, 1,3, 4-oxadiazole), thiophene, isothiazole, thiazole, thiadiazole (e.g., 1,2, 3-thiadiazole, 1,2, 4-thiadiazole, 1,3, 4-thiadiazole), pyridine, pyridazine, pyrimidine, pyrazine, triazine (e.g., 1,2, 4-triazine, 1,3, 5-triazine), and tetrazine.
In some cases, both rings of the polycyclic heteroaryl group are aromatic. Examples include indole, isoindole, indazole, benzimidazole, benzotriazole, benzofuran, benzoxazole, benzisoxazole, benzoxadiazole, benzothiophene, benzothiazole, benzisothiazole, benzothiadiazole, 1H-pyrrolo [2,3-b ] pyridine, 1H-pyrazolo [3,4-b ] pyridine, 3H-imidazo [4,5-b ] pyridine, 3H- [1,2,3] triazolo [4,5-b ] pyridine, 1H-pyrrolo [3,2-b ] pyridine, 1H-pyrazolo [4,3-b ] pyridine, 1H-imidazo [4,5-b ] pyridine, 1H- [1,2,3] triazolo [4,5-b ] pyridine, 1H-pyrrolo [2,3-c ] pyridine, 1H-pyrazolo [3,4-c ] pyridine, 3H-imidazo [4,5-c ] pyridine 3H- [1,2,3] triazolo [4,5-c ] pyridine, 1H-pyrrolo [3,2-c ] pyridine, 1H-pyrazolo [4,3-c ] pyridine, 1H-imidazo [4,5-c ] pyridine, 1H- [1,2,3] triazolo [4,5-c ] pyridine, furo [2,3-b ] pyridine, oxazolo [5,4-b ] pyridine, isoxazolo [5,4-b ] pyridine, [1,2,3] oxadiazolo [5,4-b ] pyridine, furo [3,2-b ] pyridine, oxazolo [4,5-b ] pyridine, isoxazolo [4,5-b ] pyridine, [1,2,3] oxadiazolo [4,5-b ] pyridine, furo [2,3-c ] pyridine, oxazolo [5,4-c ] pyridine, isoxazolo [1,2, 3-b ] pyridine, oxazolo [4, 4-b ] pyridine, furo [3,2-c ] pyridine, oxazolo [4,5-c ] pyridine, isoxazolo [4,5-c ] pyridine, [1,2,3] oxadiazolo [4,5-c ] pyridine, thieno [2,3-b ] pyridine, thiazolo [5,4-b ] pyridine, isothiazolo [5,4-b ] pyridine, [1,2,3] thiadiazolo [5,4-b ] pyridine, thieno [3,2-b ] pyridine, thiazolo [4,5-b ] pyridine, isothiazolo [4,5-b ] pyridine, [1,2,3] thiadiazolo [4,5-b ] pyridine, thieno [2,3-c ] pyridine, thiazolo [5,4-c ] pyridine, isothiazolo [5,4-c ] pyridine [1,2,3] thiadiazolo [5,4-c ] pyridine, thieno [3,2-c ] pyridine, thiazolo [4,5-c ] pyridine, isothiazolo [4,5-c ] pyridine, [1,2,3] thiadiazolo [4,5-c ] pyridine, quinoline, isoquinoline, cinnoline, quinazoline, quinoxaline, phthalazine, naphthyridine (e.g., 1, 8-naphthyridine, 1, 7-naphthyridine, 1, 6-naphthyridine, 1, 5-naphthyridine, 2, 7-naphthyridine, 2, 6-naphthyridine), imidazo [1,2-a ] pyridine, 1H-pyrazolo [3,4-d ] thiazole, 1H-pyrazolo [4,3-d ] thiazole, and imidazo [2,1-b ] thiazole.
In other examples, a polycyclic heteroaryl group may include a non-aromatic ring (e.g., cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl) fused to a heteroaryl ring, provided that the polycyclic heteroaryl group is bonded to the parent structure via an atom in the aromatic ring. For example, a4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-2-yl group (wherein the moiety is bonded to the parent structure via an aromatic carbon atom) is considered a heteroaryl group, while a4, 5,6, 7-tetrahydrobenzo [ d ] thiazol-5-yl group (wherein the moiety is bonded to the parent structure via a non-aromatic carbon atom) is not considered a heteroaryl group. Examples of polycyclic heteroaryl groups consisting of heteroaryl rings fused to non-aromatic rings are described below.
As used herein, the terms "comprising," "including," and "containing" are used in their open, non-limiting sense. It should also be understood that aspects and embodiments of the invention described herein may include "consist of" and/or "consist essentially of" the aspects and embodiments.
It will be understood that, whether or not the term "about" is used explicitly, each quantity given herein is intended to refer to the actual given value, and it is also intended to refer to the approximation of the given value that would reasonably be inferred based on one of ordinary skill in the art, including equivalents and approximations due to experimental and/or measurement conditions with respect to the given value.
As used herein, a "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals exposed to the dosage and concentration employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Non-limiting examples of physiologically acceptable carriers include: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants such as TWEEN TM, polyethylene glycol (PEG), and PLURONICS TM.
As used herein, the term "effective amount" or "therapeutically effective amount" of a substance is at least the minimum concentration required to cause a measurable improvement or prevention of a particular disorder. The effective amount herein may vary depending on factors such as: the disease state, age, sex and weight of the patient, and the ability of the substance to elicit a desired response in the individual. An effective amount is also an amount of any toxic or adverse effect of the treatment that is beneficial in excess of the treatment. In the case of cancer, an effective amount comprises an amount sufficient to shrink a tumor and/or reduce the rate of tumor growth (such as inhibiting tumor growth) or prevent or delay other unwanted cell proliferation in the cancer. In some embodiments, the effective amount is an amount sufficient to delay the progression of cancer. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, the effective amount is an amount sufficient to reduce the recurrence rate of the individual. An effective amount may be administered in one or more administrations. An effective amount of the drug or composition may be: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) Inhibit, delay, slow and preferably stop cancer cell infiltration into peripheral organs to some extent; (iv) Inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the appearance and/or recurrence of a tumor; (vii) Reducing the recurrence rate of the tumor, and/or (viii) alleviating to some extent one or more of the symptoms associated with the cancer. An effective amount may be administered in one or more administrations. For the purposes of this disclosure, an effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or attained in combination with one or more other agents.
"Pharmaceutical instructions" refers to instructions that are typically included in commercial packages of pharmaceutical agents that contain information about the indication, usage, dosage, administration, contraindications, other pharmaceutical agents to be combined with the packaged product, and/or warnings regarding the use of such pharmaceutical agents, and the like.
The terms "protein", "polypeptide" and "peptide" are used herein to refer to polymers having amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation to a labeling component. Typically, the proteins for use herein will have a molecular weight of at least about 5-20kDa, or at least about 20-100kDa, or at least about 100 kDa. Also included within the definition are, for example, one or more analogs (including, for example, unnatural amino acids, etc.) that contain an amino acid, as well as other modified proteins known in the art.
A "pharmaceutically acceptable salt" is a salt form that is non-toxic, biologically tolerable or otherwise biologically suitable for administration to a subject. See generally Berge et al (1977) j.pharm.sci.66,1. The particular pharmaceutically acceptable salt is a pharmacologically effective salt that is suitable for contact with the tissue of a subject without undue toxicity, irritation, or allergic response. Pharmaceutically acceptable salts include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric and the like; or acid addition salts formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid, and the like. These salts may be derived from inorganic or organic acids. Non-limiting examples of pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monobasic phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, methylsulfonate, propylsulfonate, benzenesulfonate, xylenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ -hydroxybutyrate, glycolate, tartrate and mandelate. In some embodiments, the acidic protons present in the parent compound are replaced with metal ions such as alkali metal ions, alkaline earth metal ions, or aluminum ions; or when coordinated with an organic base, forms a pharmaceutically acceptable salt. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and alkali ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylamine ethanol, tromethamine, trimethylamine, dicyclohexylamine, caffeine, procaine, sea Zhuo An, choline, betaine, ethylenediamine, glucosamine, N-ethyl-reduced glucosamine, N-methyl-reduced glucosamine, theobromine, purines, piperazines, piperidines, N-ethylpiperidines, polyamine resins, amino acids, such as lysine, arginine, histidine, and the like. Examples of pharmaceutically acceptable base addition salts include base addition salts derived from inorganic bases, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In some embodiments, the organic non-toxic base is an L-amino acid (such as L-lysine and L-arginine), bradykinin, N-ethyl reduced glucosamine, and N-methyl reduced glucosamine. Acceptable inorganic bases include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. A list of other suitable pharmaceutically acceptable salts is found in Remington's Pharmaceutical Sciences, 17 th edition, mack Publishing Company, easton, pa., 1985.
The "solvate" is formed by the interaction of a solvent with a compound. Suitable solvents include, for example, water and alcohols (e.g., ethanol). Solvates include hydrates of compounds with water in any ratio, such as monohydrate, dihydrate, and hemihydrate.
"Subject," "patient," or "individual(s)" include mammals, such as humans or other animals, and are typically human. In some embodiments, the subject (e.g., patient) to which the therapeutic agent and composition are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be of any suitable age, including infant, young, adult and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent, dog, cat, farm animal, such as a cow or horse, or the like.
The term "cancer" or "tumor" refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, invasiveness, metastatic potential, rapid growth and proliferation rate, and certain specific morphological characteristics. Cancer cells are typically in the form of solid tumors, which may be detected based on tumor masses, for example, by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and/or which may be detected due to the expression of one or more cancer-specific antigens in a sample obtained from a patient. In some embodiments, the solid tumor need not be of a measurable size. Cancer cells may also be in the form of liquid tumors, which may exist alone or be disseminated in animals. As used herein, the terms "disseminated tumor" and "liquid tumor" are used interchangeably and include, but are not limited to, leukemia and lymphoma, as well as other blood cell cancers.
The term "leukemia" refers to a type of blood or bone marrow cancer characterized by an abnormal increase in immature white blood cells (referred to as "blast cells"). Leukemia is a broad term covering a range of diseases. Which in turn is part of an even broader group of diseases affecting the blood, bone marrow and lymphatic system, all of which are known as hematological neoplasms. Leukemia can be divided into four major categories: acute lymphoblastic (or lymphoblastic) leukemia (ALL), acute myelogenous (or myelogenous or non-lymphocytic) leukemia (AML), chronic Lymphocytic Leukemia (CLL), and Chronic Myelogenous Leukemia (CML). Other leukemia types include Hairy Cell Leukemia (HCL), T-cell pre-lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia.
The term "lymphoma" refers to a group of blood cell tumors that develop from lymphocytes. Two major classes of lymphomas are Hodgkin's Lymphoma (HL) and non-hodgkin's lymphoma (NHL). Lymphomas include any lymphoid neoplasm. The main category is lymphocyte cancer, a type of white blood cell, which belongs to both the lymph and the blood and is diffuse.
As used herein, the term "cancer" includes pre-cancerous as well as malignant cancers, and also includes primary tumors (e.g., tumors whose cells have not migrated to a subject's body site other than the original tumor site) and secondary tumors (e.g., tumors arising from metastasis, migration of tumor cells to a secondary site other than the original tumor site), recurrent cancers, and refractory cancers.
The terms "cancer recurrence (cancer recurrence)" and "cancer recurrence (CANCER RELAPSE)" are used interchangeably and refer to the return of signs, symptoms, or disease after remission. Recurrent cancer cells may reproduce at the same site or another site of the primary tumor, such as in secondary cancers. Cancer cells can reproduce the same form of lesions as the primary cancer or a different form of lesions. For example, in some embodiments, the primary cancer is a solid tumor and the recurrent cancer is a liquid tumor. In other embodiments, the primary cancer is a liquid tumor and the recurrent cancer is a solid tumor. In other embodiments, the primary cancer and the recurrent cancer are both solid tumors, or both are liquid tumors. In some embodiments, the recurrent tumor expresses at least one tumor-associated antigen that is also expressed by the primary tumor.
The term "refractory cancer" as used herein refers to cancers that are not responsive to treatment, such as cancers that are resistant at the beginning of treatment (e.g., treatment with immunotherapy) or cancers that may become resistant during treatment. The term "response" refers to an anticancer response, for example in the sense that the tumor size is reduced or tumor growth is inhibited. The term may also refer to an improved prognosis, for example reflected by: an extended recurrence time, which is a period of time from a primary recurrence check or death of the second primary cancer as the first event without signs of recurrence; or increased total survival, which is the period from treatment to death of any etiology. By response or having a response is meant that a beneficial assessment indicator is achieved when exposed to a stimulus. Or negative or deleterious symptoms are minimized, alleviated, or reduced upon exposure to a stimulus. It will be appreciated that assessing the likelihood that a tumor or subject will exhibit an adverse reaction is equivalent to assessing the likelihood that a tumor or subject will not exhibit an adverse reaction (i.e., will exhibit an insufficient or no response).
As used herein, cancers include, but are not limited to, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, B cell cancer (e.g., multiple myeloma, waldenstrom's macroglobulinemiaMacrolobulinema), heavy chain diseases (such as alpha chain diseases, gamma chain diseases and mu chain diseases), benign monoclonal gammaglobulin diseases, immune cell amyloidosis, and the like. Other non-limiting examples of types of cancers suitable for use in the methods encompassed by the present invention include human sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphokaposi's sarcoma, lymphoendotheliosarcoma, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma), squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, myelogenous carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, liver carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms 'tumor (Wilms' tumor), cervical cancer, bone cancer, brain tumor, rhabdoma, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, neural tumor, astrocytoma, angioma, neuroblastoma; leukemias such as acute lymphoblastic leukemia and acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (hodgkin's and non-hodgkin's), multiple myeloma, waldenstrom's macroglobulinemia and heavy chain diseases. In some embodiments, the cancer is epithelial in nature and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers may be characterized in a variety of other ways including, but not limited to, serous, endometrium-like, mucinous, clear cells, brinz (brenner), or undifferentiated.
The term "cancer therapy" or "cancer therapeutic" as used herein refers to those therapies or agents that may exert an antitumor effect or have antitumor activity. Such anti-tumor effects or anti-tumor activity may be exhibited as a decrease in tumor cell proliferation rate, survival rate, or metastatic activity. A possible way to show anti-tumor activity is to show a decrease in the growth rate of abnormal cells generated during therapy or a stabilization or decrease in tumor size. Such activity may be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to study anti-tumor activity.
The terms "treatment" and "treatment" are intended to include alleviation or elimination of one or more of a disorder, condition, or disease, or a symptom associated with a disorder, condition, or disease; or to alleviate or eradicate the cause of the disorder, condition, or disease itself.
The terms "prevention", "prevention" and "prevention" are intended to include the following methods: postponing and/or preventing onset of the disorder, condition or disease and/or its attendant symptoms; preventing the subject from suffering from a disorder, condition, or disease; or reduce the risk of a subject suffering from a disorder, condition, or disease.
The term "substituted" means that the specified group or moiety carries one or more substituents, including but not limited to substituents such as: alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, aryl, heteroaryl, aryloxy, cyano, azido, halo, hydroxy, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl, heterocyclyl, aralkyl, sulfamoyl, sulfonylamino, sulfonyl, oxo, and the like. The term "unsubstituted" means that the specified group does not carry a substituent. When the term "substituted" is used to describe a structural system, substitution means any valency-allowed position on the system. When a group or moiety carries more than one substituent, it is understood that the substituents may be the same or different from each other. In some embodiments, a substituted group or moiety carries one to five substituents. In some embodiments, a substituted group or moiety carries one substituent. In some embodiments, a substituted group or moiety carries two substituents. In some embodiments, a substituted group or moiety carries three substituents. In some embodiments, a substituted group or moiety carries four substituents. In some embodiments, a substituted group or moiety carries five substituents.
"Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" encompasses both "alkyl" and "substituted alkyl" as defined herein. It will be appreciated by those skilled in the art that for any group containing one or more substituents, the group is not intended to introduce any substitution or pattern of substitution that is sterically impractical, synthetically infeasible, and/or unstable per se. It is also understood that where a group or moiety is optionally substituted, the invention includes embodiments wherein the group or moiety is substituted as well as embodiments wherein the group or moiety is unsubstituted.
As used herein, the term "Q tag" refers to a portion of a polypeptide containing a glutamine residue that includes a side chain modified to include an amide bound to a compound, after a transglutaminase-mediated reaction with a compound containing-NH 2 amine, to provide a conjugate containing the polypeptide portion. Q tags are known in the art. In some embodiments, the Q tag is attached to the C-terminus of the heavy chain of the antibody. In some embodiments, the Q tag is attached to the light chain of the antibody. In some embodiments, the Q tag is naturally occurring. For example, mutations in N297 to N297A expose Q295 of the antibody, where conjugation may occur (numbered according to EU index, e.g., as listed in Edelman, G.M. et al, proc.Natl. Acad.USA,63,78-85 (1969) and Kabat, E.A. et al, sequences of proteins of immunological insert 5 th edition-USDepartment of HEALTH AND Human Services, NIH publication No. 91-3242, pages 662, 680, 689 (1991)). In some embodiments, the Q tag is within the Fc domain of the antibody.
Conjugates of
Immunostimulatory oligonucleotides have been used in a variety of therapeutic applications. To improve targeting specificity and in vivo distribution, an immunomodulatory oligonucleotide (e.g., cpG ODN) may be conjugated to a targeting moiety (e.g., a polypeptide, such as a SIRP-a antibody). In particular, transglutaminase mediated reactions are useful for carrying out such conjugation reactions due to their high reaction rates and suitable site specificity. The present disclosure provides polypeptide-oligonucleotide conjugates (such as SIRP-a antibody-oligonucleotide conjugates) that exhibit advantageous activity.
The present invention is based at least in part on the following findings: the anti-SIRP-alpha antibody-CpG oligonucleotide conjugates are effective in activating human bone marrow cells (e.g., monocytes and dendritic cells), stimulating cytokine production, and mediating anti-tumor activity against SIRP-alpha positive and SIRP-alpha negative tumor cells. These conjugates integrate TLR9 activation and blocking of CD 47-SIRP-alpha interaction with bone marrow cells such that the anti-tumor immune response comes from both the innate and adaptive immune systems. Thus, the present invention provides oligonucleotide-SIRP-a antibody conjugates with robust manufacturability and strong activity in various preclinical models.
Provided herein are oligonucleotide-SIRP-a antibody conjugates (i.e., SIRP-a antibody-oligonucleotide conjugates; SIRP-a antibody conjugates; anti-SIRP-a antibody-CpG oligonucleotide conjugates) in which the oligonucleotide and SIRP-a antibody are linked together via a linking moiety. In some embodiments, one SIRP-a antibody may be conjugated to one or more oligonucleotides. In some embodiments, the oligonucleotide-antibody conjugate is a conjugate comprising a SIRP-a antibody or antigen-binding fragment thereof and one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment is linked to one or more Q tag peptides (Q) comprising at least one glutamine residue, wherein each immunomodulatory oligonucleotide is linked to a Q tag peptide via an amide bond to a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof;
wherein:
indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
Each Q is independently a Q tag peptide sequence comprising at least one glutamine residue;
each L is independently a bond or a linker moiety linked to Q via an amide bond with a glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide.
In some embodiments, the conjugate is a conjugate comprising a SIRP-alpha antibody or antigen binding fragment thereof and one or more immunomodulatory oligonucleotides (P), wherein the SIRP-alpha antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47), wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (A),
Or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof;
wherein:
indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
Each Q independently comprises a Q tag peptide comprising peptide sequence RPQGF (SEQ ID NO: 47);
each L is independently a bond or a linker moiety linked to Q via an amide bond with a glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide.
In other embodiments, the conjugate is a conjugate comprising a SIRP-alpha antibody or antigen binding fragment thereof and one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen binding fragment is linked to one or more Q-tag peptides (Q) comprising at least one glutamine residue, wherein each immunomodulatory oligonucleotide is linked to the Q-tag peptide via an amide bond to the glutamine residue of the Q-tag peptide and a linker (L), as shown in formula (A),
Or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof;
wherein:
indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
each Q is independently a Q tag peptide comprising at least one glutamine residue;
each L is independently a bond or a linker moiety linked to Q via an amide bond with a glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide selected from the group consisting of the oligonucleotides of table 15.
In one embodiment, the DAR of the oligonucleotide-SIRP-a antibody conjugate is in the range of about 1 to about 20, about 1 to about 10, about 1 to about 8, about 1 to about 4, or about 1 to about 2. In another embodiment, the DAR of the oligonucleotide-SIRP-a antibody conjugate is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
In some embodiments, the conjugate comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more Q tag peptides. In some embodiments, the conjugate comprises one, two, three, four, five, six, seven, eight, nine, ten, or twenty Q tag peptides. In some embodiments, the conjugate has 2Q tag peptides. In some embodiments, the conjugate comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more immunomodulatory oligonucleotides. In some embodiments, the conjugate comprises one, two, three, four, five, six, seven, eight, nine, ten, or twenty immunomodulatory oligonucleotides. In some embodiments, the conjugate has an immunomodulatory oligonucleotide. Exemplary conjugates are shown in fig. 9A-9D.
Immunomodulatory oligonucleotides
In one aspect, the oligonucleotide in the oligonucleotide-SIRP-a antibody conjugate is an immunomodulatory (e.g., immunostimulatory) oligonucleotide. In certain embodiments, the immunomodulatory oligonucleotide comprises a 5-modified uridine or a 5-modified cytidine. In certain embodiments, including a 5-modified uridine (e.g., 5-ethynyl-uridine) at the 5 'end of the immunomodulatory oligonucleotide (e.g., among two 5' end nucleosides) can enhance the immunomodulatory properties of the oligonucleotide. In certain embodiments, the immunomodulatory oligonucleotide is shorter than a typical CpG ODN of 18 to 28 nucleotides in length (e.g., comprising a total of about 6 to about 16 nucleotides or about 12 to about 14 nucleotides). In certain embodiments, shorter immunomodulatory oligonucleotides (e.g., those comprising a total of about 6 to about 16 nucleotides or about 12 to about 14 nucleotides) retain the immunomodulatory activity of longer typical CpG ODNs compared to longer CpG ODNs; or exhibit higher immunomodulatory activity (e.g., as measured by nfkb activation or by a change in the level of expression of an activated or functional cell surface marker such as CD40, HLADR, CD69 or CD80 or by a change in the level of at least one cytokine (e.g., IL-6 or IL-10). In certain embodiments, the immunomodulatory oligonucleotide comprises an abasic spacer. In certain embodiments, the immunomodulatory oligonucleotide comprises an internucleoside phosphotriester.
In certain embodiments, the immunomodulatory oligonucleotides provided herein exhibit stability (e.g., stability against nucleases) that is superior to CpG ODNs that contain predominantly internucleoside phosphates (e.g., greater than 50% internucleoside phosphates) without substantially sacrificing their immunostimulatory activity. This can be accomplished, for example, by incorporating at least 50% (e.g., at least 70%) of the internucleoside phosphorothioate or phosphorodithioate or via inclusion of internucleoside phosphotriesters and/or internucleoside abasic spacers. Phosphotriesters and abasic spacers are also suitable for conjugation to targeting moieties. Phosphate-based phosphotriesters and abasic spacers can also be used to reduce off-target activity relative to oligonucleotides having a complete phosphorothioate backbone. Without wishing to be bound by theory, this effect may be achieved by reducing self-delivery without disrupting targeting moiety-mediated delivery to the target cells. Thus, an oligonucleotide provided herein can include about 15 or fewer, about 14 or fewer, about 13 or fewer, about 12 or fewer, about 11 or fewer, or about 10 or fewer consecutive internucleoside phosphorothioates. For example, an immunostimulatory oligonucleotide comprising a total of about 12 to about 16 nucleosides may contain about 10 or fewer consecutive internucleoside phosphorothioates.
The immunostimulatory oligonucleotides provided herein may contain a total of about 50 or fewer, about 30 or fewer, about 28 or fewer, or about 16 or fewer nucleosides. The immunostimulatory oligonucleotide may contain a total of at least 6, about 10 or more, or about 12 or more nucleosides. For example, the immunostimulatory oligonucleotide may contain a total of about 6 to about 30, about 6 to about 28, about 6 to about 20, about 6 to about 16, about 10 to about 20, about 10 to about 16, about 12 to about 28, about 12 to about 20, or about 12 to about 16 nucleosides.
In certain embodiments, the immunostimulatory oligonucleotide comprises one or more phosphotriesters (e.g., internucleoside phosphotriesters) and/or phosphorothioates (e.g., about 1 to about 6 or about 1 to about 4), e.g., at one or both termini (e.g., within six 5 'terminal nucleosides or six 3' terminal nucleosides). The inclusion of one or more internucleoside phosphate esters and/or phosphorothioates may enhance the stability of the oligonucleotide by reducing the rate of exonuclease mediated degradation.
In certain embodiments, the immunostimulatory oligonucleotide comprises a phosphotriester or terminal phosphodiester, wherein the phosphotriester or terminal phosphodiester comprises a linker bound to a targeting moiety or conjugate group and optionally bound to one or more (e.g., about 1 to about 6) auxiliary moieties. In certain embodiments, the immunostimulatory oligonucleotide comprises only one linker. In certain embodiments, the immunostimulatory oligonucleotide comprises only one conjugate group.
The oligonucleotides provided herein may be hybridization oligonucleotides comprising one strand and its partial or complete complementary sequence. The hybridization oligonucleotide can have at least 6 complementary base pairs (e.g., about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or about 23) up to the total number of nucleotides present in the shorter strand involved. For example, the hybridization portion of the hybridization oligonucleotide can contain about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or about 23 base pairs.
In one aspect, the oligonucleotide in the oligonucleotide-SIRP-a antibody conjugate comprises one or more CpG sites. In some embodiments, the oligonucleotide comprises at least 1, at least 2, or at least 3 CpG sites. In some embodiments, the oligonucleotide is an antisense oligonucleotide. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2' -hydroxy nucleotide). In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to: deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2 '-modified nucleotides, 3' -to 3 '-linked (inverted) nucleotides, nucleotides comprising unnatural bases, bridged nucleotides, peptide Nucleic Acids (PNAs), 2',3 '-broken nucleotide mimics (unlocking nucleobase analogues), locked nucleotides, 3' -O-methoxy (2 'internucleoside linked) nucleotides, 2' -F-arabinonucleotides, 5'-Me, 2' -fluoro nucleotides, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides and cyclopropyl phosphonate containing nucleotides (cPrpN). 2' -modified nucleotides (i.e., nucleotides having groups other than hydroxyl groups at the 2' position of the five-membered sugar ring) include, but are not limited to, 2' -O-alkyl nucleotides, 2' -deoxy-2 ' -halo nucleotides, 2' -deoxy nucleotides, 2' -methoxyethyl (2 ' -O-2-methoxyethyl) nucleotides, 2' -amino nucleotides, 2' -aminoalkyl nucleotides, and 2' -alkyl nucleotides. In some embodiments, the modified nucleotide is selected from the group consisting of: 5-bromo-2 '-O-methyluridine, 5-bromo-2' -deoxyuridine, 2 '-O-methyluridine, 2' -deoxyuridine, 2 '-O-methylthymidine, 2' -O-methylcytidine, 2'-O- (2-methoxyethyl) thymidine and 8-oxo-7, 8-dihydro-2' -deoxyguanosine. Not all positions of a given compound need be uniformly modified. Conversely, more than one modification may be incorporated in a single oligonucleotide or even in a single nucleotide thereof. Oligonucleotides may be synthesized and/or modified by methods known in the art. The modification at one nucleotide is independent of the modification at another nucleotide.
Modified nucleobases include synthetic and natural nucleobases such as 5-substituted pyrimidine, 6-aza-pyrimidine and N-2, N-6 and O-6 substituted purine (e.g. 2-aminopropyl adenine, 5-propynyluracil or 5-propynylcytosine), 5-methylcytosine (5-Me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g. 6-methyl, 6-ethyl, 6-isopropyl or 6-N-butyl) derivatives of adenine and guanine, 2-alkyl (e.g. 2-methyl, 2-ethyl, 2-isopropyl or 2-N-butyl) derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halogenoalkyluracil (e.g. 5-bromouracil and 5-iodouracil), cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azo uracil, 6-azo-cytosine, 6-azo-thymine, 5-uracil, 8-hydroxy (8-halo-8-hydroxy-, 8-amino-, 8-halo-8-hydroxy-, 8-halo-8-amino-, 8-halo-hydroxy-8-amino-8-halo-substituted adenine and other alkyl derivatives, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanines and 7-methyladenines, 8-azaguanines and 8-azaadenines, 7-deazaguanines, 7-deazaadenines, 3-deazaguanines and 3-deazaadenines.
In some embodiments, one or more nucleotides of an oligonucleotide are linked by a non-standard linkage or backbone (e.g., a modified internucleoside linkage or modified backbone). In some embodiments, the modified internucleoside linkage is a non-phosphate containing covalent internucleoside linkage. Modified internucleoside linkages or backbones include, but are not limited to, 5' phosphorothioate groups, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonate or 3' -alkylene phosphonate), chiral phosphonates, phosphonites, phosphoramidates (e.g., 3' -phosphoramidate, aminoalkyl phosphoramidate or thiocarbonyl phosphoramidate), thiocarbonylalkyl-phosphonate, thiocarbonylalkyl phosphotriesters, morpholino linkages, borane phosphates with normal 3' -5' linkages, 2' -5' linkage analogues of borane phosphates, or borane phosphates with reversed polarity, wherein pairs of adjacent nucleoside units link 3' -5' to 5' -3' or 2' -5' to 5' -2'. In some embodiments, the modified internucleoside linkage or backbone does not have a phosphorus atom. Modified internucleoside linkages without a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatom or heterocyclic intersugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, thioether backbones, sulfoxide backbones, sulfone backbones, formyl and thioformyl backbones, methyleneformyl and thioformyl backbones, olefin-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S and CH 2 components.
In some embodiments, the oligonucleotide comprises 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, at least 13, at least 14, or at least 15 phosphorothioate linkages. In some embodiments, the oligonucleotide comprises 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, at least 13, at least 14, or at least 15 phosphorodithioate linkages. In some embodiments, the oligonucleotide comprises 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 phosphorothioate linkages. In some embodiments, the oligonucleotide comprises 1, 2,3, 4,5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 phosphorodithioate linkages. In some embodiments, phosphorothioate internucleoside linkages or phosphorodithioate internucleoside linkages are located between nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, 18-20 or 19-21 from the 5' end of the oligonucleotide. In some embodiments, the oligonucleotide comprises one or more modified nucleotides and one or more modified internucleoside linkages.
In some embodiments, the oligonucleotide comprises an end cap. In some embodiments, the terminal cap is at the 3' end of the oligonucleotide. In some embodiments, the terminal cap is at the 5' end of the oligonucleotide. In some embodiments, the end caps are at the 5 'and 3' ends of the oligonucleotides. The term "end cap" may also be referred to as a "cap" and has a meaning commonly accepted in the art. For example, the term refers to a moiety, which may be a chemically modified nucleotide or non-nucleotide that may incorporate one or more ends of a nucleic acid molecule of the invention. These terminal modifications may protect the nucleic acid molecule from exonuclease degradation and may aid in intracellular delivery and/or localization. In a non-limiting example, the cap includes, but is not limited to: a polymer; a ligand; locked Nucleic Acid (LNA); a glyceryl group; an abasic ribose residue; reverse deoxygenation of abasic residues; an inverted nucleotide; 4',5' -methylene nucleotide; 1- (β -D-erythrofuranosyl) nucleotide; a 5' -hydrosulfide moiety; 4' -thio nucleotide; carbocyclic nucleotides; 1, 5-anhydrohexitol nucleotides; l-nucleotides; an alpha-nucleotide; modified base nucleotides; dithiophosphate linkages; a threo-pentofuranosyl nucleotide; acyclic 3',4' -seco nucleotide; acyclic 3, 4-dihydroxybutyl nucleotides; acyclic 3, 5-dihydroxypentyl nucleotides; a 3'-3' -inverted nucleotide moiety; a 3'-3' -inverted abasic moiety; a 3'-2' -inverted nucleotide moiety; a 3'-2' -inverted abasic moiety; 1, 4-butanediol phosphate; 3' -phosphoramidates; hexyl phosphate; amino hexyl phosphate; 3' -phosphate; 3' -phosphorothioate; a 5'-5' -inverted nucleotide moiety; a 5'-5' -inverted abasic moiety; 5' -phosphoramidate; 5' -phosphorothioate; 1, 4-butanediol phosphate; a 5' -amino group; bridged and/or unbridged 5' -phosphoramidates; phosphorothioate and/or phosphorodithioate; or a bridged or unbridged methylphosphonate moiety. In some embodiments, the oligonucleotide comprises one or more end cap molecules. In some embodiments, [ N ] is a 3' end cap. In some embodiments, the 3' end cap is O- (3-hydroxypropyl) phosphorothioate.
In some embodiments, the length of the oligonucleotide is about 10-30, about 10-15, about 15-20, about 20-25, about 25-30, about 15-25 nucleotides. In some embodiments, the oligonucleotide is about 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
In another aspect, the oligonucleotide of the conjugate comprises:
Wherein the method comprises the steps of
B and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5;
Indicating the point of attachment of the immunomodulatory oligonucleotide P to the remainder of the conjugate;
X 5' is a structure A 5' terminal nucleoside of (2); /(I)
X 3' is a structure3' Terminal nucleoside of (2);
Y PTE is a compound having a structure Wherein represents the point of attachment to the rest of the oligonucleotide, and/>Represents the point of attachment to the linker L, or if L is absent,/>Represents the point of attachment to the Q tag peptide Q via an amide bond at the glutamine residue;
y 3' is a compound having a structure Is a terminal phosphotriester of (a);
each X N is independently a compound having a structure A nucleoside of (2);
Each Y N is independently a compound having a structure Is a nucleoside linker; wherein each B N is independently a modified or unmodified nucleobase;
Each R N is independently-H or-O-C 1-4 alkyl, wherein the C 1-4 alkyl of said-O-C 1-4 alkyl is optionally further substituted with-O-C 1-4 alkyl;
b 5' and B 3' are independently modified or unmodified nucleobases;
R 5' and R 3' are independently-H or-O-C 1-C4 alkyl, wherein the C 1-4 alkyl of the-O-C 1-4 alkyl is optionally further substituted with-O-C 1-4 alkyl;
each T 1 is independently O or S;
Each T 2 is independently O - or S -; and
T 3 is a group comprising an oligoethylene glycol moiety; and
R 1 is C 1-4 alkylene-hydroxy.
In certain embodiments, the oligonucleotide comprises a nucleotide having a modified nucleobase. In some embodiments, B 5' is a modified nucleobase. In other embodiments, B 3' is a modified nucleobase. In some embodiments, B 5' is an unmodified nucleobase. In other embodiments, B 3' is an unmodified nucleobase. In other embodiments, at least one B N is a modified nucleobase.
In certain embodiments, b is an integer in the range of about 1 to about 15. In certain embodiments, b is an integer of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In certain embodiments, b is an integer of about 3, about 4, about 11, or about 14. In certain embodiments, b is an integer of about 3. In certain embodiments, b is an integer of about 4. In certain embodiments, b is an integer of about 11. In certain embodiments, b is an integer of about 14.
In certain embodiments, c is an integer in the range of about 0 to about 10. In certain embodiments, c is an integer of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. In certain embodiments, c is an integer of about 0 or about 8. In certain embodiments, c is an integer of about 0. In certain embodiments, c is an integer of about 8.
In certain embodiments, b is an integer of about 3, and c is an integer of about 8. In certain embodiments, b is an integer of about 4, and c is an integer of about 8. In certain embodiments, b is an integer of about 11, and c is an integer of about 0. In certain embodiments, b is an integer of about 14, and c is an integer of about 0.
In certain embodiments, b and c together range from about 5 to about 20. In certain embodiments, b and c together range from about 5 to about 15. In certain embodiments, b and c together are about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In certain embodiments, b and c together are about 8, about 9, about 10, about 11, about 12, about 13, or about 14. In certain embodiments, b and c together are about 11. In certain embodiments, b and c together are about 12. In certain embodiments, b and c together are about 14.
In certain embodiments, each X N is independently a 2 '-deoxyribonucleoside or a 2' -modified ribonucleoside. In certain embodiments, each X N is independently 2 '-deoxyadenosine (a), 2' -deoxyguanosine (G), 2 '-deoxycytidine (C), 5-halo-2' -deoxycytidine, 2 '-deoxythymidine (T), 2' -deoxyuridine (U), 5-halo-2 '-deoxyuridine, 2' -fluororibonucleoside, 2 '-methoxyribonucleoside, or 2' - (2-methoxyethoxy) ribonucleoside. In certain embodiments, each X N is independently a 2' -deoxyribonucleoside. In certain embodiments, each X N is independently 2' -deoxyadenosine, 2' -deoxyguanosine, 2' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2' -deoxythymidine, 2' -deoxyuridine, or 5-halo-2 ' -deoxyuridine. In certain embodiments, each X N is independently 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 2' -deoxythymidine, 5-bromo-2 '-deoxyuridine, or 5-iodo-2' -deoxyuridine.
In certain embodiments, X 3' is a2 '-deoxyribonucleoside or a 2' -modified ribonucleoside. In certain embodiments, X 3' is 2' -deoxyribonucleoside. In certain embodiments, X 3' is 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 5-halo-2' -deoxycytidine, 2 '-deoxythymidine, 2' -deoxyuridine, 5-halo-2 '-deoxyuridine, 2' -fluororibonucleoside, 2 '-methoxyribonucleoside, or 2' - (2-methoxyethoxy) ribonucleoside. In certain embodiments, X 3' is 2' -deoxyadenosine, 2' -deoxyguanosine, 2' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2' -deoxythymidine, 2' -deoxyuridine, or 5-halo-2 ' -deoxyuridine. In certain embodiments, X 3' is 2' -deoxythymidine. In certain embodiments, X 3' is a 2' -deoxyribonucleoside having a substituted pyrimidine base. In certain embodiments, X 3' is a 2' -deoxyribonucleoside having a 5-substituted pyrimidine base. In certain embodiments, X 3' is 2' -deoxythymidine, 5-halo-2 ' -deoxycytidine, or 5-halo-2 ' -deoxyuridine. In certain embodiments, X 3' is 2' -deoxythymidine, 5-bromo-2 ' -deoxycytidine, 5-iodo-2 ' -deoxycytidine, 5-bromo-2 ' -deoxyuridine, or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X 3' is 2' -deoxythymidine, 5-bromo-2 ' -deoxyuridine, or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X 3' is a terminal nucleotide comprising a 3' end capping group. In certain embodiments, the 3' end capping group is a terminal phosphate. In certain embodiments, the 3' end capping group is 3-hydroxy-propylphosphoryl (i.e., -P (O 2)-OCH2CH2CH2 OH).
In certain embodiments, X 5' is a2 '-deoxyribonucleoside or a 2' -modified ribonucleoside. In certain embodiments, X 5' is 2' -deoxyribonucleoside. In certain embodiments, X 5' is 2 '-deoxyadenosine, 2' -deoxyguanosine, 2 '-deoxycytidine, 5-halo-2' -deoxycytidine, 2 '-deoxythymidine, 2' -deoxyuridine, 5-halo-2 '-deoxyuridine, 2' -fluororibonucleoside, 2 '-methoxyribonucleoside, or 2' - (2-methoxyethoxy) ribonucleoside. In certain embodiments, X 5' is 2' -deoxyadenosine, 2' -deoxyguanosine, 2' -deoxycytidine, 5-halo-2 ' -deoxycytidine, 2' -deoxythymidine, 2' -deoxyuridine, or 5-halo-2 ' -deoxyuridine. In certain embodiments, X 5' is a 2' -deoxyribonucleoside having a substituted pyrimidine base. In certain embodiments, X 5' is a 2' -deoxyribonucleoside having a 5-substituted pyrimidine base. In certain embodiments, X 5' is 2' -deoxythymidine, 5-halo-2 ' -deoxycytidine, or 5-halo-2 ' -deoxyuridine. In certain embodiments, X 5' is 5-halo-2' -deoxycytidine. In some embodiments, X 5' is 2 '-deoxyuridine, 5-halo-2' -deoxyuridine, 2 '-methoxyuridine, or 5-halo-2' -methoxyuridine. In certain embodiments, X 5' is 5-halo-2' -deoxyuridine. In certain other embodiments, X 5' is 2' -deoxyuridine. In certain embodiments, X 5' is 5-halo-2' -methoxyuridine. In certain other embodiments, X 5' is 2' -methoxyuridine. In certain embodiments, X 5' is 2' -deoxythymidine, 5-bromo-2 ' -deoxycytidine, 5-iodo-2 ' -deoxycytidine, 5-bromo-2 ' -deoxyuridine, or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X 5' is 2' -deoxythymidine, 5-bromo-2 ' -deoxyuridine, or 5-iodo-2 ' -deoxyuridine. In certain embodiments, X 5' is 5-bromo-2' -deoxyuridine. In certain embodiments, X 5' is 5-iodo-2' -deoxyuridine. In certain embodiments, X 5' has a 3' -phosphorothioate group. In certain embodiments, X 5' has a chiral 3' -phosphorothioate group with Rp. In certain embodiments, X 5' has a 3' -phosphorothioate group with the chirality of Sp.
In certain embodiments, Y PTE is an internucleoside phosphorothioate triester.
In some embodiments, Y PTE is Wherein Z is O or S; d is an integer in the range of about 0 to about 50; two on the right side of the structure/>Indicating the point of attachment to oligonucleotide P; and/>, on the left side of the structureIndicating the point of attachment to the remainder of the conjugate. In certain embodiments, Z is O. In certain embodiments, Z is S. In certain embodiments, d is an integer in the range of about 0 to about 10. In certain embodiments, d is an integer in the range of about 0 to about 5. In certain embodiments, d is an integer of about 0, about 1, about 2, about 3, about 4, or about 5. In certain embodiments, d is an integer of about 0, about 1, or about 3.
In some embodiments, Y PTE isWherein Z is O or S; d is an integer in the range of about 0 to about 50; two on the right side of the structure/>Indicating the point of attachment to oligonucleotide P; and to the left of the structureIndicating the point of attachment to the remainder of the conjugate. In certain embodiments, Z is O. In certain embodiments, Z is S. In certain embodiments, d is an integer in the range of about 0 to about 10. In certain embodiments, d is an integer in the range of about 0 to about 5. In certain embodiments, d is an integer of about 0, about 1, about 2, about 3, about 4, or about 5. In certain embodiments, d is an integer of about 0, about 1, or about 3.
In certain embodiments, the oligonucleotide comprises one additional internucleoside phosphotriester. In one embodiment, the additional internucleoside phosphate is a C 1-6 alkyl phosphate. In another embodiment, the additional internucleoside phosphate is ethyl phosphate.
In certain embodiments, the oligonucleotide comprises a 5-halo-2' -deoxyuridine. In one embodiment, the 5-halo-2 '-deoxyuridine is 5-fluoro-2' -deoxyuridine, 5-bromo-2 '-deoxyuridine, or 5-iodo-2' -deoxyuridine. In another embodiment, the 5-halo-2 ' -deoxyuridine is 5-bromo-2 ' -deoxyuridine or 5-iodo-2 ' -deoxyuridine. In yet another embodiment, the 5-halo-2 '-deoxyuridine is 5-fluoro-2' -deoxyuridine. In yet another embodiment, the 5-halo-2 '-deoxyuridine is 5-bromo-2' -deoxyuridine. In yet another embodiment, the 5-halo-2 '-deoxyuridine is 5-iodo-2' -deoxyuridine.
In certain embodiments, the oligonucleotide comprises three or more 2' -deoxycytidines. In certain embodiments, the oligonucleotide comprises three 2' -deoxycytidines.
In certain embodiments, the oligonucleotide comprises four or more 2' -deoxyguanosine. In certain embodiments, the oligonucleotide comprises four 2' -deoxyguanosine.
In certain embodiments, the oligonucleotide comprises three 2 '-deoxycytidine and four 2' -deoxyguanosine. In certain embodiments, the oligonucleotide comprises one, two, or three CG dinucleotides. In certain embodiments, the oligonucleotide comprises three CG dinucleotides.
In certain embodiments, the oligonucleotide comprises three or more 2' -deoxythymidine. In certain embodiments, the oligonucleotide comprises three, four, five, six, seven, or eight 2' -deoxythymidine. In certain embodiments, the oligonucleotide comprises three, four, five, or eight 2' -deoxythymidine.
In certain embodiments, the oligonucleotide does not comprise 2' -deoxyadenosine. In certain embodiments, the oligonucleotide comprises one or two 2' -deoxyadenosines.
In certain embodiments, the length of the oligonucleotide is in the range of about 5 to about 20 or about 6 to about 15. In certain embodiments, the length of the oligonucleotide is about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In certain embodiments, the length of the oligonucleotide is about 10, about 11, about 12, about 13, about 14, or about 15.
In certain embodiments, the oligonucleotide comprises one or more internucleoside phosphorothioates. In certain embodiments, all of the internucleoside phosphates in the oligonucleotide are internucleoside phosphorothioates. In certain embodiments, the oligonucleotide comprises one or more chiral internucleoside phosphorothioates.
In certain embodiments, an oligonucleotide comprising the sequence of N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174) or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof, as described for example in WO2018/189382 A1.
In one embodiment, the oligonucleotide comprises the sequence of N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), or a stereoisomer, a mixture of two or more diastereomers, a tautomer, or a mixture of two or more tautomers thereof; or a pharmaceutically acceptable salt, solvate or hydrate thereof; wherein:
x is an integer in the range of about 1 to about 4;
N 1 is absent or 2' -deoxythymidine;
N 2 is a 2' -deoxyribonucleotide with a modified nucleobase;
n 3 is 2' -deoxyadenosine or 2' -deoxythymidine, each optionally comprising a 3' -phosphotriester;
N 4 is 2 '-deoxyadenosine or 2' -deoxythymidine;
n 5 is 2 '-deoxythymidine, optionally comprising 3' -phosphotriester; and
C is 2 '-deoxynucleoside and G is 2' -deoxyguanosine.
In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), x is an integer of about 1, about 2, about 3, or about 4. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), x is an integer of about 1. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), x is an integer of about 4.
In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 1 is absent. In certain embodiments, in N 1N2CGN3CG(T)x GN4CGN5 T (SEQ ID NO: 174), N 1 is 2' -deoxythymidine.
In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 2 is a 2' -deoxyribonucleotide with a substituted pyrimidine base. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 2 is a 2' -deoxyribonucleotide having a 5-substituted pyrimidine base. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 2 is 5-halo-2 '-deoxycytidine or 5-halo-2' -deoxyuridine. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 2 is 5-bromo-2 '-deoxyuridine or 5-iodo-2' -deoxyuridine.
In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 3 is 2 '-deoxyadenosine comprising a 3' -phosphotriester. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 3 is 2' -deoxythymidine. In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 3 is 2 '-deoxythymidine comprising a 3' -phosphotriester.
In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 4 is 2' -deoxyadenosine. In certain embodiments, in N 1N2CGN3CG(T)x GN4CGN5 T (SEQ ID NO: 174), N 4 is 2' -deoxythymidine.
In certain embodiments, in N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174), N 5 is 2' -oxythymidine. In certain embodiments, in N 1N2CGN3CG(T)x GN4CGN5 T (SEQ ID NO: 174), N 5 is 2 '-deoxythymidine comprising a 3' -phosphotriester.
In certain embodiments, the oligonucleotide of N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174) comprises one or more internucleoside phosphorothioates or phosphorodithioates. In certain embodiments, the oligonucleotide of N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174) comprises at least one chiral internucleoside phosphorothioate or phosphorodithioate. In certain embodiments, the oligonucleotide of N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174) comprises at least one chiral dithiophosphate. In certain embodiments, the oligonucleotide of N 1N2CGN3CG(T)xGN4CGN5 T (SEQ ID NO: 174) is an oligonucleotide sequence as described, for example, in WO2018/189382 A1.
With respect to the characters used herein (e.g., with respect to 5 'and/or 3' identification), it should be noted that the characters 'may appear in different fonts herein, e.g., a Times font (') is used herein and a different font (') is used when used as part of a chemical formula, but the meaning of the characters' is intended to be the same, independent of font.
In certain embodiments, the oligonucleotide is an immunostimulatory oligonucleotide. In certain embodiments, the oligonucleotides provided herein function as PAMSs. In certain embodiments, the oligonucleotides provided herein activate an innate immune response or stimulate an adaptive immune response by triggering TLR9 signaling. In certain embodiments, the oligonucleotides provided herein are TLR9 agonists.
In certain embodiments, the oligonucleotide is a CpG oligonucleotide comprising modifications comprising 5-halouridine or 5-alkynyluridine, or truncated versions thereof (e.g., those comprising a total of about 6 to about 16 nucleosides). In certain embodiments, a truncated oligonucleotide provided herein comprises a truncated oligonucleotide sequence from which one or more of the 3' terminal nucleotides are eliminated or one or more of the nucleotides within the sequence are excised.
In certain embodiments, the oligonucleotide comprises at least one immunostimulatory sequence (ISS). In certain embodiments, the oligonucleotides provided herein comprise about 1, about 2, about 3, or about 4 ISS. ISS in immunostimulatory oligonucleotides depends on the targeted organism. A common feature of ISS for use in the oligonucleotides provided herein is the cytidine-p-guanosine sequence, wherein p is an internucleoside phosphate diester (e.g., phosphate or phosphorothioate) or an internucleoside phosphate triester. In certain embodiments, cytidine and guanosine in the ISS each independently comprise 2' -deoxyribose. In certain embodiments, the oligonucleotides provided herein comprise about 1, about 2, or about 3 human ISS. In certain embodiments, the human ISS is CG or NCG, wherein N is uridine, cytidine, or thymidine, or a modified uridine or cytidine; and G is guanosine or a modified guanosine. In certain embodiments, the modified uridine or cytidine is 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 5-alkynyluridine (e.g., 5-ethynyluridine or 5-propynyluridine), 5-heteroaryluridine, or 5-halocytidine. In certain embodiments, the modified guanosine is 7-deazaguanosine. In certain embodiments, the human ISS is NCG, and in one embodiment, N is 5-halouridine. In certain embodiments, the human ISS is UCG, in one embodiment, U is 5-alkynyluridine, and in another embodiment, U is 5-ethynyluridine. In certain embodiments, the human targeted oligonucleotides provided herein comprise an ISS within four consecutive nucleotides comprising a 5' terminal nucleotide. In certain embodiments, the human targeted oligonucleotides provided herein comprise a 5' terminal ISS. In certain embodiments, the oligonucleotides provided herein comprise murine ISS. In certain embodiments, the murine ISS is a hexamer nucleotide sequence: pu-Pu-CG-Py-Py, wherein each Pu is independently a purine nucleotide and each Py is independently a pyrimidine nucleotide.
In certain embodiments, the 5 '-flanking nucleotides relative to CpG in the oligonucleotides provided herein are free of 2' -alkoxyribose. In certain embodiments, the 5 '-flanking nucleotides relative to CpG in the oligonucleotides provided herein comprise only 2' -deoxyribose as a sugar.
In certain embodiments, the oligonucleotide has: (1) High levels of phosphorothioate or phosphorodithioate (e.g., at least 50%, at least 60%, at least 70% or at least 80% of the nucleosides can be linked by phosphorothioate or phosphorodithioate); (2) the absence of poly-G tails; (3) The nucleosides in the oligonucleotides comprise 2 '-deoxyribose or 2' -modified ribose (e.g., 2 '-halo (e.g., 2' -fluoro, 2 '-bromo, or 2' -iodo) or optionally substituted 2 '-alkoxy (e.g., 2' -methoxy)); and/or (4) comprises a 5' -terminal ISS, i.e., NCG, wherein N is uridine, cytidine, or thymidine, or a modified uridine or cytidine, and G is guanosine or a modified guanosine.
In certain embodiments, the oligonucleotides inhibit the adaptive immune response by reducing activation of TLR9 signaling (e.g., via TLR9 antagonism). In certain embodiments, the immunosuppressive oligonucleotides provided herein comprise at least two 2 '-alkoxyribonucleotides that are 5' -pendant with respect to CpG, as described by the formula N 1-N2 -CG, wherein N 1 and N 2 are each independently a2 '-alkoxyribose (e.g., 2' -methoxyribose) containing nucleotide.
In some embodiments, the oligonucleotide has the following structure:
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is O - or S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
U 5' is-H or halogen;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, provided that if one R g1、Rg2、Rg3 and R g4 are oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy;
r 1 is C 1-4 alkylene-hydroxy;
r 2 is-H or methyl; and
N is an integer from 0 to 2.
In other embodiments, the oligonucleotide has the following structure:
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is O - or S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy;
r 1 is C 1-4 alkylene-hydroxy;
r 2 is-H or methyl; and
N is an integer from 0 to 2.
In other embodiments, the oligonucleotide has the following structure:
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is O - or S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, provided that if one R g1、Rg2、Rg3 and R g4 are oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy;
r 1 is C 1-4 alkylene-hydroxy;
r 2 is-H or methyl; and
N is an integer from 0 to 2.
In some embodiments, the oligonucleotide comprises one or more of the unmodified sequences that differ from the sequences shown in table 1 by 0, 1, 2, or 3 nucleobases. In some embodiments, the oligonucleotide comprises one or more of a modified sequence differing from the sequence shown in table 2 by 0, 1, 2, or 3 nucleobases.
TABLE 1 unmodified oligonucleotides
TABLE 2 modified oligonucleotides
/>
/>
* U: 5-bromo-2' -deoxyuridine
G: 8-oxo-7, 8-dihydro-2' -deoxyguanosine
U: 5-bromo 2' -OMe uridine
C:2' -OMe-cytidine
T:2' -OMe-Thymidine
U:2' -OMe-uridine
U:2' -deoxyuridine
T:2' -OMOE thymidine
Ts: triester phosphate linker-PEG after thymidine 24-NH2
Ts: after thymidineA phosphotriester linker;
lowercase letters: 2' -deoxynucleotides
S: phosphorothioate linkages
S2: dithiophosphate linkages
c3：
s2-c3：
In some embodiments, the oligonucleotide is functionalized with a chemical tag to attach to the linking moiety. In some embodiments, the chemical tag is linked to an internucleoside linkage of the oligonucleotide. In some embodiments, the chemical tag is linked to a 5' internucleoside linkage. In some embodiments, the chemical tag is linked to a 3' internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage. In some embodiments, the internucleoside linkage is a phosphorodithioate linkage. In some embodiments, the chemical tag is closer to the 5 'end than the 3' end of the oligonucleotide. In some embodiments, the chemical tag is attached to a nucleobase.
Connection part
In another aspect, the oligonucleotide is conjugated to the SIRP-a antibody via a linking moiety. The length, rigidity and chemical composition of the linking moiety affect the rate of the conjugation reaction and the stability of the resulting conjugate. In some embodiments, the linking moiety comprises polyethylene glycol (PEG). In some embodiments, the PEG contains about 10-50 ethylene glycol units. In some embodiments, the linking moiety is an aliphatic chain.
For formula (a), the linking moiety is represented by L. In some embodiments, the linker L comprises an oligo glycol or polyethylene glycol moiety. In certain embodiments, the linker L is of a structureWherein/>Indicates the point of attachment to Y PTE, and/>Indicating the point of attachment to the remainder of the conjugate.
In other embodiments, the linker L is of a structureWherein/>Indicates the point of attachment to Y PTE, and/>Indicating the point of attachment to the remainder of the conjugate. In some embodiments, L 1 is absent. In some embodiments, L 1 is unsubstituted alkyl. In some embodiments, L 1 is independently unsubstituted C 1-6 alkyl. In some embodiments, each L 1 is methyl or ethyl. In some embodiments, L 1 is independently substituted alkyl. In some embodiments, L 1 is independently substituted C 1-6 alkyl. In some embodiments, L 1 is C 1-6 alkyl substituted with one or more substituents selected from the group consisting of: alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxy, nitro, carboxyl, thiol, sulfanyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo.
In some embodiments, L 2 is absent. In some embodiments, L 2 is unsubstituted or substituted alkyl.
In some embodiments, L 3 is absent. In some embodiments, L 3 is a linker moiety. In some embodiments, the linker moiety is an unsubstituted or substituted alkyl group. In some embodiments, the linker moiety is independently unsubstituted C 1-6 alkyl. In some embodiments, the linker moiety is methyl or ethyl. In some embodiments, the linker moiety is independently a substituted alkyl group. In some embodiments, the linker moiety is independently a substituted C 1-6 alkyl group. In some embodiments, the linker moiety is a C 1-6 alkyl group substituted with one or more substituents selected from the group consisting of: alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano, azido, halo, hydroxy, nitro, carboxyl, thiol, sulfanyl, alkyl, alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyl, and oxo. In some embodiments, the linker moiety is an amino acid residue. In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, glutamic acid, and proline. In some embodiments, the linker is methyl. In some embodiments, the linker moiety is-R 5C(O)R6NHR7 -, where R 5 and R 7 are independently absent or unsubstituted or substituted alkyl, and R 6 is an amino acid residue. In some embodiments, the amino acid is selected from the group consisting of glycine, alanine, glutamic acid, and proline. In some embodiments, the linker moiety is-R 3C(O)NHR4 -, where R 3 and R 4 are independently absent or unsubstituted or substituted alkyl. In some embodiments, R 3 is methylene and R 4 is- (CH 2)4 -. In some embodiments, R 3 is methylene and R 4 is absent when the oligonucleotide is more than one (i.e., p=2), the two L 1 may be different or the same, the two L 2 may be different or the same, and the two L 3 may be different or the same.
In some embodiments, m is about 3-10, about 10-15, about 15-20, about 20-25, about 25-30, about 5-16, about 15-30, about 15-25, or about 20-30. In some embodiments, m is 20, 21, 22, 23, 24, or 25.
Anti-SIRP-alpha antibodies and Q-tag peptides
In various embodiments, antibodies that specifically bind to SIRP-a (i.e., anti-SIRP-a antibodies, SIRP-a targeting antibodies), particularly antibodies that specifically bind to human SIRP-a, and conjugates thereof are described herein. In some embodiments, the antibody binds to a human SIRP-a polypeptide (e.g., an extracellular domain of a human SIRP-a polypeptide, such as a D1 domain). In some embodiments, SIRP-a refers to human SIRP-a, and the antibody specifically binds to human SIRP-a. SIRP-a gene and polypeptide sequences (e.g., human gene and polypeptide sequences) are known in the art (see exemplary sequences below). In some embodiments, the SIRP-a conjugate (i.e., an anti-SIRP-a conjugate) specifically binds to a cell (e.g., a bone marrow cell or a tumor cell) that expresses a SIRP-a polypeptide (e.g., a human SIRP-a polypeptide) on its cell surface. Human SIRP-alpha is also known as BIT, MFR, P, SIRP, MYD-1, SHPS1, CD172A and PTPNS1. In some embodiments, a human SIRP-a polypeptide refers to a polypeptide encoded by a human SIRPA gene, for example as described by NCBI reference Seq ID No. 140885.
In some embodiments, the SIRP-a antibody is selected from the group consisting of: polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, chimeric antibodies, and antibody fragments. In some embodiments, the SIRP-a antibody is, for example, a full length antibody comprising an Fc region (including, but not limited to, the exemplary Fc regions described herein). In some embodiments, the SIRP-a antibody fragment is selected from the group consisting of: fab, fab '-SH, F (ab') 2, fv fragments, scFv, single domain antibodies, single heavy chain antibodies, and single light chain antibodies. In some embodiments, the SIRP-a antibody fragment is selected from the group consisting of: fab, fab '-SH, F (ab') 2, fv fragments and scFv fragments.
Any of the anti-SIRP-a antibodies described herein may be used in any of the conjugates of the disclosure. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises a heavy chain Variable (VH) domain that comprises a CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), a CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57), and a CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58). In some embodiments, a SIRP-alpha antibody of the present disclosure comprises a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60), and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61). In some embodiments, a SIRP-a antibody of the disclosure comprises: a heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58); and a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61).
In some embodiments, a SIRP-a antibody of the disclosure comprises a VH domain comprising the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKG LEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62). In some embodiments, a SIRP-a antibody of the disclosure comprises VL domains ：SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVT LIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYD QSSYTNPFGGGTQLTVL(SEQ ID NO:63)、SYELTQPPSVSVSPGQT ARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSG SSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTKLTVL(SEQ ID NO:64) and SYELTQPPSVSVSPGQTARITCSGGSYSSYYYA WYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQA EDEADYYCGGYDQSSYTNPFGGGTELTVL(SEQ ID NO:65). comprising an amino acid sequence selected from the group consisting of SEQ ID No. 62 and a VL domain comprising an amino acid sequence of SEQ ID No. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO. 62 and a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises a VH domain comprising the amino acid sequence of SEQ ID NO. 62 and a VL domain comprising the amino acid sequence of SEQ ID NO. 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises an antibody VH domain (e.g., comprising the amino acid sequence of SEQ ID NO: 62) and an Fc region. In some embodiments, a SIRP-a antibody (including a C-terminal Q tag peptide) of the present disclosure comprises an antibody heavy chain comprising an amino acid sequence selected from the group consisting of:
EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGRPQGFGPP(SEQ ID NO:66)、EVQLVESGGGVVQPGGSLRLSC AASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP(SEQ ID NO:67) And EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP(SEQ ID NO:68). in some embodiments, a SIRP-a antibody of the disclosure comprises an antibody heavy chain comprising an amino acid sequence selected from the group consisting of:
EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:87)、EVQLVESGGGVVQPGGSLRLSCAASGFTF SSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:88) And EVQLVESGGGVVQP GGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:89). in some embodiments, a SIRP-a antibody of the present disclosure comprises an antibody heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 87-89 and a Q tag peptide or sequence of the present disclosure.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63, 64 or 65. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises the amino acid sequence of SEQ ID NO. 87. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63, 64 or 65. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 87 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises the amino acid sequence of SEQ ID NO. 67. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63, 64 or 65. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises the amino acid sequence of SEQ ID NO. 88. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63, 64 or 65. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 88 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises the amino acid sequence of SEQ ID NO. 68. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63, 64 or 65. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain that comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 89 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63, 64 or 65. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 89 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 63. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 89 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO. 64. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:89 and an antibody light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 65.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain (e.g., comprising the amino acid sequence of SEQ ID NO:63, 64 or 65) and an antibody constant light Chain (CL) domain. In some embodiments, the CL domain is a kappa CL domain (e.g., a human kappa CL domain). In some embodiments, the CL domain is a λcl domain (e.g., a human λcl domain). In some embodiments, the CL domain is the human IGLC or IGLC domain. In some embodiments, a SIRP-a antibody of the disclosure comprises an antibody light chain ：GQPKANPTVTLFPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNN KYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:69)、GQPKANPTVTLFPPSSEELQANKATLVCLISD FYPGAVTVAWKADGSPVKAGVETTKPSKQSSDKYAASSYLSLTP EQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:70) or comprising the amino acid sequence of GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS(SEQ ID NO:71).
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO:63, 64 or 65 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO:69, 70 or 71. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 63 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 69. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 63 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 70. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 63 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 71. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 64 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 69. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 64 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 70. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 64 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 71. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain that comprises an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 65 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 69. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain that comprises an antibody VL domain comprising the amino acid sequence of SEQ ID NO. 65 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO. 70. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody light chain comprising an antibody VL domain comprising the amino acid sequence of SEQ ID NO:65 and an antibody CL domain comprising the amino acid sequence of SEQ ID NO: 71.
In some embodiments, a SIRP-a antibody of the present disclosure comprises an antibody light chain ：SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTQLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:72)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:73)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTELTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:74)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTQLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSSDKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:75)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSSDKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:76)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTELTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSSDKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:77)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTQLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:78)、SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:79) comprising an amino acid sequence selected from the group consisting of SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTELTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:80).
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 77. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 or 87 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 80.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 72. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 74. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 76. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 77. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 78. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 or 88 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 80.
In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 72. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 74. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 76. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 77. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 78. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 or 89 and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 80.
In some embodiments, the SIRP-a antibody further comprises at least one Q tag peptide sequence. In some embodiments, at least one Q tag is attached to the heavy chain of a SIRP-a antibody. In certain embodiments, at least one Q tag is fused to the C-terminus of the heavy chain of a SIRP-a antibody. In other embodiments, at least one Q tag is attached to the light chain of a SIRP-a antibody. In other embodiments, at least one Q tag is within the Fc domain or Fc region. Exemplary and non-limiting Q tag peptide sequences are disclosed herein and can be used in any of the antibodies or conjugates of the present disclosure.
In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to an extracellular domain (e.g., D1 domain) of a human SIRP- αv1 polypeptide and/or a human SIRP- αv2 polypeptide. In some embodiments, the human SIRP- αv1 polypeptide comprises the amino acid sequence of EEELQVIQPD KSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEG HFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPD DVEFKSGAGTELSVRAKPS(SEQ ID NO:81). In some embodiments, the human SIRP- αv2 polypeptide comprises the amino acid sequence of EEELQVIQPDKSVSVAAGESA ILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTK RENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVR AKPS(SEQ ID NO:82). In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to the extracellular domain (e.g., D1 domain) of a human SIRP- αv1 polypeptide and a human SIRP- αv2 polypeptide. In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to a human SIRP- αv1 polypeptide and/or an extracellular domain (e.g., D1 domain) of a human SIRP- αv2 polypeptide with a dissociation constant (K D) of less than 100nM, less than 10nM, less than 1nM, or 1pM or less.
In some embodiments, an antibody or conjugate of the disclosure binds to an extracellular domain (e.g., D1 domain) of a monkey SIRP-a polypeptide (e.g., D1 domain of a monkey SIRP-a polypeptide). In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to an extracellular domain (e.g., D1 domain) of a cynomolgus monkey SIRP-a polypeptide (e.g., found in an organism cynomolgus monkey (Macaca fascicularis)). In some embodiments, the cynomolgus monkey SIRP-a polypeptide comprises the amino acid sequence of EEELQVIQPEKSVSVAAGESATLNCTATSLIPVGPIQWFRGVG PGRELIYHQKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTY YCVKFRKGSPDVELKSGAGTELSVRAKPS(SEQ ID NO:84). In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to an extracellular domain (e.g., a D1 domain) of a mouse SIRP-a polypeptide (e.g., a D1 domain of a mouse SIRP-a polypeptide). In some embodiments, the mouse SIRP-a polypeptide comprises the amino acid sequence of KELKVTQPEKSVSVAAGDSTVLNCTLTSLLPVGPIKWYRGVGQSR LLIYSFTGEHFPRVTNVSDATKRNNMDFSIRISNVTPEDAGTYYCV KFQKGPSEPDTEIQSGGGTEVYVLAKPS(SEQ ID NO:83).
In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to an extracellular domain (e.g., D1 domain) of a human SIRP- β polypeptide and/or a human SIRP- γ polypeptide. In some embodiments, a human SIRP- β polypeptide refers to a polypeptide encoded by a human SIRPB gene, e.g., as described by NCBI Ref Seq ID No. 10326. In some embodiments, the human SIRP-gamma polypeptide refers to a polypeptide encoded by a human SIRPG gene, for example, as described by NCBI Ref Seq ID No. 55423. In some embodiments, the human SIRP- β polypeptide comprises the amino acid sequence of EDELQVIQPEKSVSVAAGESATLRCAMTSLIPVGPIMWFRGAGAG RELIYNQKEGHFPRVTTVSELTKRNNLDFSISISNITPADAGTYYCV KFRKGSPDDVEFKSGAGTELSVRAKPS(SEQ ID NO:85). In some embodiments, the human SIRP-gamma polypeptide comprises the amino acid sequence of EEELQMIQPEKLLL VTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPR VTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFK SGPGTEMALGAKPS(SEQ ID NO:86).
In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to an extracellular domain (e.g., D1 domain) of a human SIRP- αv1 polypeptide, a human SIRP- αv2 polypeptide, a cynomolgus monkey SIRP- α polypeptide, a mouse SIRP- α polypeptide, a human SIRP- β polypeptide, and/or a human SIRP- γ polypeptide. In some embodiments, an antibody or conjugate of the disclosure binds or is capable of binding to an extracellular domain (e.g., D1 domain) of a human SIRP- αv1 polypeptide, a human SIRP- αv2 polypeptide, a cynomolgus monkey SIRP- α polypeptide, a mouse SIRP- α polypeptide, a human SIRP- β polypeptide, and a human SIRP- γ polypeptide. Cross-reactivity across mammalian species (such as human, monkey, and mouse) may be advantageous for preclinical testing of antibodies or conjugates of the disclosure, for example.
In some embodiments, an antibody or conjugate of the disclosure modulates SIRP-a signaling in a cell expressing a human SIRP-a polypeptide. In some embodiments, an antibody or conjugate of the disclosure antagonizes SIRP-a signaling in a cell expressing a human SIRP-a polypeptide. In some embodiments, an antibody or conjugate of the disclosure interferes with SIRP-a signaling in a cell expressing a human SIRP-a polypeptide. In some embodiments, an antibody or conjugate of the disclosure agonizes SIRP-a signaling in cells expressing a human SIRP-a polypeptide. In some embodiments, SIRP-a signaling includes one or more intracellular signaling events mediated by activation of a SIRP-a polypeptide, including, but not limited to, tyrosine phosphorylation of an intracellular region of SIRP-a, phosphatase (e.g., SHP 1) binding, adapter protein binding (e.g., SCAP2, FYB, and/or GRB 2), and nitric oxide production. Various assays to measure SIRP-a signaling in cells include, but are not limited to, SIRP-a phosphorylation, SHP1 and SHP2 co-immunoprecipitation, PI3 kinase signaling, cytokine production (inflammatory IL-12, IL-23, tnfα, IFN and inhibitory cytokines IL-10, IL-4, IL-13, cell surface marker levels of M1 and M2 macrophage markers) or dendritic cell activation and function; kharitonenkov, A. Et al (1997) Nature 386:181-6; ochi, F. Et al (1997) biochem. Biophys. Res. Commun.239:483-7; kim, E.J. et al (2013) Inflammation Research 62:377-86; yi, T.et al (2015) Immunity 43:764-75.
In some embodiments, the cell expressing the human SIRP-a polypeptide is a leukocyte. In some embodiments, the cell is a macrophage, dendritic cell, neutrophil, eosinophil, or myeloid-derived suppressor cell (MDSC). In some embodiments, an antibody or conjugate of the disclosure reduces or antagonizes SIRP-a signaling in a cell expressing a human SIRP-a polypeptide by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., using one or more of the SIRP-a signaling assays described herein or otherwise known in the art. In some embodiments, an antibody or conjugate of the disclosure increases or agonizes SIRP-a signaling in a cell expressing a human SIRP-a polypeptide by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, for example, using one or more of the SIRP-a signaling assays described herein or otherwise known in the art.
In some embodiments, an antibody or conjugate of the disclosure modulates an intercellular phenotype mediated by SIRP-a. In some embodiments, the antibodies or conjugates of the disclosure enhance phagocytosis of macrophages expressing human SIRP-a polypeptides. For example, the phagocytic activity of a macrophage treated with or contacted with an antibody or conjugate of the present disclosure may be compared to the phagocytic activity of a macrophage not treated with or contacted with an antibody or conjugate, or the phagocytic activity of a macrophage expressing a human SIRP-a polypeptide and treated with or contacted with an antibody or conjugate of the present disclosure may be compared to the phagocytic activity of a macrophage not expressing a human SIRP-a polypeptide and treated with or contacted with an antibody or conjugate. Exemplary phagocytosis assays can be found, for example, in Wieskopf, K.et al (2013) Science 341:88 and WILLINGHAM, S.B. et al (2012) Proc.Natl. Acad.Sci.109:6662-7. In some embodiments, an antibody or conjugate of the disclosure increases phagocytosis of macrophages expressing a human SIRP-a polypeptide by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, for example using one or more of the phagocytosis assays described herein or otherwise known in the art.
In some embodiments, an antibody or conjugate of the disclosure enhances activation of dendritic cells expressing a human SIRP-a polypeptide (e.g., increases the degree of activation of individual dendritic cells, or increases the proportion of activated dendritic cells within a sample population). For example, activation of dendritic cells treated with or contacted with an antibody or conjugate of the present disclosure may be compared to activation of dendritic cells not treated with or contacted with an antibody or conjugate, or activation of dendritic cells expressing a human SIRP-a polypeptide and treated with or contacted with an antibody or conjugate of the present disclosure may be compared to activation of dendritic cells not expressing a human SIRP-a polypeptide and treated with or contacted with an antibody or conjugate. Exemplary dendritic cell activation assays are described herein. In some embodiments, an antibody or conjugate of the disclosure increases dendritic cell (e.g., expression of a human SIRP-a polypeptide) activation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., using one or more of the dendritic cell activation assays described herein or otherwise known in the art.
In some embodiments, an antibody or conjugate of the disclosure inhibits the growth of a tumor expressing CD47 (e.g., human CD 47) in vivo. For example, in vivo growth of tumors expressing CD47 and treated with an antibody or conjugate of the present disclosure can be compared to in vivo growth of tumors expressing CD47 and not treated with an antibody or conjugate of the present disclosure. Exemplary in vivo tumor growth assays are described herein. In some embodiments, an antibody or conjugate of the disclosure inhibits in vivo growth of a tumor expressing CD47 by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., using one or more of the in vivo tumor growth assays described herein or otherwise known in the art.
In some embodiments, an antibody or conjugate of the disclosure inhibits in vivo growth of a tumor that expresses SIRP-a (e.g., human SIRP-a). For example, the in vivo growth of tumors that express SIRP- α and are treated with an antibody or conjugate of the present disclosure can be compared to the in vivo growth of tumors that express SIRP- α and are not treated with an antibody or conjugate of the present disclosure. Exemplary in vivo tumor growth assays are described herein. In some embodiments, an antibody or conjugate of the disclosure inhibits in vivo growth of a tumor that expresses SIRP-a by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, e.g., using one or more of the in vivo tumor growth assays described herein or otherwise known in the art.
In some embodiments, an antibody or conjugate of the disclosure blocks binding between the extracellular domain (e.g., D1 domain) of a human SIRP-a polypeptide and a human CD47 polypeptide (e.g., igSF domain of a human CD47 polypeptide). For example, an antibody or conjugate and CD47 polypeptide may "compete" for the same SIRP-a epitope, and/or an antibody or conjugate that binds to SIRP-a may be mutually exclusive to CD47 that binds to SIRP-a. The binding interface between SIRP-a and CD47 is known for residues of two proteins involved in binding; see HATHERLEY, D.et al (2007) J.biol.chem.282:14567-75 and Nakaishi, A.et al (2008) J.mol.biol.375:650-60. Exemplary assays for determining whether an antibody or conjugate blocks the binding of CD47 to SIRP-a are known in the art. In some embodiments, an antibody or conjugate of the disclosure blocks binding between the extracellular domain (e.g., D1 domain) of a human SIRP-a polypeptide and the IgSF domain of a human CD47 polypeptide in an in vitro assay, e.g., using purified SIRP-a and/or CD47 polypeptides. In some embodiments, a "blocking" anti-SIRP-alpha antibody or conjugate of the present disclosure binds to an extracellular domain (e.g., D1 domain) of a SIRP-alpha polypeptide at one or more amino acid positions that also bind to CD47 in the CD47 SIRP-alpha complex. In some embodiments, an antibody or conjugate of the disclosure blocks binding between an extracellular domain (e.g., D1 domain) of a human SIRP-a polypeptide expressed on a first cell surface and an IgSF domain of a human CD47 polypeptide expressed on a second cell surface, e.g., an in vivo binding assay between polypeptides expressed on a cell surface. In some embodiments, an in vivo assay may assess binding between the extracellular domain (e.g., D1 domain) of a human SIRP-a polypeptide expressed on a first cell surface and the IgSF domain of a human CD47 polypeptide expressed on a second cell surface by assaying one or more aspects of SIRP-a signaling, such as one or more intracellular signaling events mediated by activation of the SIRP-a polypeptide, including, but not limited to, tyrosine phosphorylation of the intracellular region of SIRP-a, phosphatase (e.g., SHP 1) binding, adapter protein binding (e.g., SCAP2, FYB and/or GRB 2), cytokine production (e.g., IL-10, IL-1 β, IFN, or TNF), and nitric oxide production; and/or one or more intercellular phenotypes including, but not limited to, macrophage phagocytosis and other activation or inhibition phenotypes of macrophages, neutrophils, dendritic cells, eosinophils, and myeloid-derived suppressor cells (MDSCs).
In some embodiments that may be combined with any of the preceding embodiments, the SIRP-a antibody or conjugate comprises an Fc region. In certain embodiments, the Fc region is a human Fc region selected from the group consisting of: an IgG1 Fc region, an IgG2 Fc region, and an IgG4 Fc region. In some embodiments, the Fc region is a wild-type human IgG1, igG2, or IgG4 Fc region. In some embodiments, the Fc region is a human Fc region comprising one or more amino acid substitutions that reduce or eliminate one or more effector functions as compared to the effector functions of a human Fc region lacking the amino acid substitutions. In some embodiments, the Fc region is a human IgG4 Fc region comprising an S228P substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the Fc region is a human IgG1 Fc region substitution comprising L234A, L a and G237A substitutions, the amino acid positions being numbered according to the EU index. In other embodiments, the Fc region is: (a) A human IgG1 Fc region comprising an L234A, L a and/or G237A substitution, the amino acid positions being numbered according to the EU index; (b) A human IgG2 Fc region comprising a330S and/or P331S substitution, amino acid positions numbered according to the EU index; (c) A human IgG4 Fc region comprising S228P and/or L235E substitutions, the amino acid positions being numbered according to the EU index; (d) A human IgG1 Fc region comprising an N297A substitution, amino acid positions numbered according to the EU index; (e) A human IgG1 Fc region comprising a D265A substitution, the amino acid positions being numbered according to the EU index; or (f) a human IgG2 Fc region comprising an N297A substitution, the amino acid positions being numbered according to the EU index. In some embodiments, the Fc region is a human Fc region comprising one or more amino acid substitutions that reduce or eliminate binding to human C1q as compared to binding of a human Fc region lacking the amino acid substitutions. In some embodiments, the Fc region is a human Fc region comprising one or more amino acid substitutions that reduce or eliminate ADCC as compared to Antibody Dependent Cellular Cytotoxicity (ADCC) of a human Fc region lacking amino acid substitutions. Exemplary Fc regions are provided in the antibody heavy chain sequences disclosed herein.
Antibodies to target cell surface antigens can trigger immunostimulatory and effector functions associated with Fc receptor (FcR) engagement on immune cells. There are a variety of Fc receptors specific for a particular class of antibodies, including IgG (gamma receptor), igE (eta receptor), igA (alpha receptor), and IgM (mu receptor). Binding of the Fc region to Fc receptors on the cell surface can trigger a variety of biological responses including phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity or ADCC) and inflammatory mediator release, placental transfer and control of immunoglobulin production. In addition, binding of the C1 component of complement to antibodies activates the complement system. Complement activation can be important for the lysis of cellular pathogens. However, complement activation can also stimulate inflammatory responses and can also be associated with autoimmune allergies or other immune disorders. Variant Fc regions with reduced or eliminated ability to bind to certain Fc receptors are useful in the production of therapeutic antibodies and Fc fusion polypeptide constructs that function by targeting, activating or neutralizing ligand functions without damaging or disrupting local cells or tissues. In some embodiments, the Fc region has wild-type or native CDC activity.
In some embodiments, an Fc domain may refer to a dimer having two Fc domain monomers. In the wild-type Fc domain, the two Fc domain monomers dimerize by interacting between the two CH3 antibody constant domains and by forming one or more disulfide bonds between the hinge domains of the two dimerizing Fc domain monomers. In some embodiments, the Fc domain is mutated to lack effector function, e.g., a "dead Fc domain". In some embodiments, each of the Fc domain monomers in the Fc domain includes amino acid substitutions in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and an Fc receptor, such as fcγ receptor (fcγr), fcα receptor (fcαr), or fcepsilon (fcepsilon R).
The Fc domain is not directly involved in binding of an antibody to its target, but may be involved in various effector functions, such as antibody involvement in antibody-dependent cytotoxicity. In some embodiments, the Fc domain in an antibody or conjugate of the present disclosure comprises one or more amino acid substitutions, additions or insertions, deletions, or any combination thereof that result in reduced effector functions, such as reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced complement-dependent cell solubility (CDC) and reduced antibody-dependent cell-mediated phagocytosis (ADCP), or any combination thereof. In some embodiments, the antibodies or conjugates of the disclosure are characterized by reduced binding (e.g., minimal or no binding) to human Fc receptor and reduced binding (e.g., minimal or no binding) to complement protein C1 q. In some embodiments, the antibodies or conjugates of the disclosure are characterized by reduced binding (e.g., minimal binding or no binding) to human fcyri, fcyriia, fcyriib, fcyriiib, or any combination thereof and C1 q. To alter or reduce antibody-dependent effector functions, such as ADCC, CDC, ADCP or any combination thereof, in some embodiments, the Fc domain in an antibody or conjugate of the disclosure is of the IgG class and comprises one or more amino acid substitutions at E233, L234, L235, G236, G237, D265, D270, N297, E318, K320, K322, a327, a330, P331, or P329 (EU index numbering according to Kabat (Sequences of Proteins of Immunological Interest, 5 th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991))).
In some embodiments, a SIRP-a antibody or conjugate comprising a non-native Fc region described herein exhibits reduced or eliminated binding to at least one of the fcγ receptors CD16a, CD32b, CD32c, and CD64 as compared to a polypeptide construct comprising a native Fc region. In some cases, a SIRP-a antibody or conjugate described herein exhibits reduced or eliminated binding to CD16a, CD32b, CD32c, and CD64 fcγ receptors.
CDC refers to a cytotoxic form in which the complement cascade is activated by complement component C1q binding to antibody Fc. In some embodiments, a SIRP-a antibody or conjugate comprising a non-native Fc region described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in C1q binding compared to an antibody or conjugate comprising a wild-type Fc region. In some cases, an antibody or conjugate comprising a non-native Fc region as described herein exhibits reduced CDC as compared to an antibody or conjugate comprising a wild-type Fc region. In some embodiments, an antibody or conjugate comprising a non-native Fc region as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in CDC compared to an antibody or conjugate comprising a wild-type Fc region. In some cases, antibodies or conjugates comprising a non-native Fc variant as described herein exhibit negligible CDC compared to antibodies or conjugates comprising a wild-type Fc region.
In some embodiments, the Fc variants herein are minimally glycosylated or have a reduced degree of glycosylation relative to the wild-type sequence. In some embodiments, deglycosylation is achieved via an N297A mutation or by mutating N297 to any amino acid other than N. In some embodiments, deglycosylation is achieved by disrupting the motifs N-Xaa1-Xaa2-Xaa3 (SEQ ID NO: 175), wherein N = asparagine; xaa1 = any amino acid other than P (proline); xaa2=t (threonine), S (serine), or C (cysteine); and xaa3=any amino acid other than P (proline). In one embodiment, the N-Xaa1-Xaa2-Xaa3 (SEQ ID NO: 175) motif refers to residues 297-300 as specified according to Kabat et al, 1991. In some embodiments, the mutation of any one or more of N, xaa, xaa2, or Xaa3 deglycosylates the Fc variant.
In some embodiments, variants of the antibody IgG constant region (e.g., fc variants) possess reduced ability to specifically bind fcγ receptors or have reduced ability to induce phagocytosis. In some embodiments, variants of the antibody IgG constant region (e.g., fc variants) possess reduced ability to specifically bind fcγ receptors and have reduced ability to induce phagocytosis. For example, in some embodiments, the Fc domain is mutated to lack effector function, typically a "dead" Fc domain. For example, in some embodiments, the Fc domain includes specific amino acid substitutions known to minimize interactions between the Fc domain and fcγ receptor. In some embodiments, the Fc domain monomer is from an IgG1 antibody and comprises one or more of the amino acid substitutions L234A, L235A, G237A and N297A (specified according to the EU numbering system according to Kabat et al, 1991). In some embodiments, one or more additional mutations are included in such IgG1Fc variants. Non-limiting examples of such additional mutations of human IgG1Fc variants include E318A and K322A. In some examples, human IgG1Fc variants have up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer mutations in total compared to the wild-type human IgG1 sequence. In some embodiments, one or more additional deletions are included in such IgG1Fc variants. For example, in some embodiments, the C-terminal lysine of the Fc IgG1 heavy chain constant region is deleted, e.g., to increase the homogeneity of the polypeptide when the polypeptide is produced in a bacterial or mammalian cell. In some cases, human IgG1Fc variants have up to 12, 11, 10, 9, 8, 7, 6, 5, or 4 or fewer deletions in total compared to the wild-type human IgG1 sequence.
In some embodiments, the Fc domain monomer is from an IgG2 or IgG4 antibody and comprises the amino acid substitution a330S, P S, or both a330S and P331S. The aforementioned amino acid positions are defined according to Kabat et al (1991). For a given antibody, the Kabat numbering of amino acid residues may be determined by alignment with a "standard" Kabat numbering sequence in the homologous region of the antibody sequence. In some embodiments, the Fc variant comprises a human IgG2 Fc sequence comprising one or more of the a330S, P S and N297A amino acid substitutions specified according to the EU numbering system according to Kabat et al (1991). In some embodiments, one or more additional mutations are included in such IgG2 Fc variants. Non-limiting examples of such additional mutations of human IgG2 Fc variants include V234A, G237A, P238S, V L and H268A (specified according to the EU numbering system according to Kabat et al (1991)). In some examples, human IgG2 Fc variants have up to 12, 11, 10, 9, 8, 7, 6,5, 4, 3, or fewer mutations in total compared to the wild-type human IgG2 sequence. In some embodiments, one or more additional deletions are included in such IgG2 Fc variants. For example, in some embodiments, the C-terminal lysine of the Fc IgG2 heavy chain constant region is deleted, e.g., to increase the homogeneity of the polypeptide when the polypeptide is produced in a bacterial or mammalian cell. In some examples, human IgG2 Fc variants have up to 12, 11, 10, 9, 8, 7, 6,5, or 4 or fewer deletions in total compared to the wild-type human IgG2 sequence.
When the Fc variant is an IgG4 Fc variant, in some embodiments, such Fc variant comprises an S228P mutation (specified according to Kabat et al (1991)), such as represented in SEQ ID NO:104 in Table 7. In some cases, human IgG4 Fc variants have up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations in total compared to the wild-type human IgG4 sequence.
In some embodiments, the Fc variant comprises at least one of mutations L234A, L235A, G a or N297A of the IgG1 Fc region or at least one of mutations a330S, P331S or N297A of the IgG2 Fc region. In some embodiments, the Fc variant comprises at least two of mutations L234A, L235A, G a or N297A of the IgG1 Fc region or at least two of mutations a330S, P331S or N297A of the IgG2 Fc region. In some embodiments, the Fc variant comprises at least three of mutations L234A, L235A, G a or N297A of the IgG1 Fc region or consists of mutations a330S, P331S and N297A of the IgG2 Fc region. In some embodiments, the Fc variant consists of mutations L234A, L235A, G a and N297A. In some embodiments, the Fc variant comprises mutations L234A, L a and G237A (IgG 1 AAA) of the IgG1 Fc region.
In some embodiments, the Fc variant exhibits reduced binding to an Fc receptor of a subject as compared to a wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits abrogated binding to the Fc receptor of the subject as compared to the wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits reduced phagocytosis compared to a wild-type human IgG Fc region. In some embodiments, the Fc variant exhibits eliminated phagocytosis compared to the wild-type human IgG Fc region.
Antibody-dependent cell-mediated cytotoxicity, also referred to herein as ADCC, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells, such as Natural Killer (NK) cells and neutrophils, thereby enabling these cytotoxic effector cells to specifically bind to and subsequently kill antigen-bearing target cells. Antibody-dependent cell-mediated phagocytosis, also referred to herein as ADCP, refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain phagocytes (e.g., macrophages), thereby enabling these phagocytic effector cells to specifically bind to antigen-bearing target cells and subsequently phagocytose and digest the target cells. Ligand-specific high affinity IgG antibodies directed against the surface of target cells can stimulate cytotoxic or phagocytic cells and can be used for such killing. In some embodiments, an antibody or conjugate comprising an Fc variant as described herein exhibits reduced ADCC or ADCP as compared to an antibody or conjugate comprising a wild-type Fc region. In some embodiments, an antibody or conjugate comprising an Fc variant as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in ADCC or ADCP as compared to an antibody or conjugate comprising a wild-type Fc region. In some embodiments, an antibody or conjugate comprising an Fc variant as described herein exhibits eliminated ADCC or ADCP as compared to an antibody or conjugate comprising a wild-type Fc region.
Complement-directed cytotoxicity, also referred to herein as CDC, refers to a form of cytotoxicity in which the complement cascade is activated by complement component C1q bound to the Fc of an antibody. In some embodiments, an antibody or conjugate comprising an Fc variant as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in C1q binding compared to an antibody or conjugate comprising a wild-type Fc region. In some cases, an antibody or conjugate comprising an Fc variant as described herein exhibits reduced CDC as compared to an antibody or conjugate comprising a wild-type Fc region. In some embodiments, an antibody or conjugate comprising an Fc variant as described herein exhibits at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in CDC compared to an antibody or conjugate comprising a wild-type Fc region. In some cases, antibodies or conjugates comprising an Fc variant as described herein exhibit negligible CDC compared to antibodies or conjugates comprising a wild-type Fc region.
Fc variants herein include variants that exhibit reduced binding to fcγ receptors compared to the wild-type human IgG Fc region. For example, in some embodiments, the Fc variant exhibits less binding to fcγ receptors than the wild-type human IgG Fc region, as described in the examples. In some examples, binding of the Fc variant to the fcγ receptor is reduced by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (complete elimination of effector function). In some embodiments, the reduced binding is to any one or more fcγ receptors, e.g., CD16a, CD32b, CD32c, or CD64.
In some examples, the Fc variants disclosed herein exhibit reduced phagocytosis compared to their wild-type human IgG Fc regions. Such Fc variants exhibit reduced phagocytosis compared to their wild-type human IgG Fc region, wherein phagocytosis activity is reduced by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the factor. In some examples, the Fc variant exhibits abrogated phagocytosis compared to its wild-type human IgG Fc region.
In some embodiments, the Q tag of the present disclosure is linked to a SIRP-a antibody. In some embodiments, the oligonucleotides of the disclosure are conjugated to a SIRP-a antibody via one or more Q tags. In some embodiments, the Q tag comprises a glutamine residue attached to the remainder of the conjugate. In other embodiments of aspects of the invention that may be combined with any of the embodiments herein, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NOS 39-55. In some embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of the peptide sequences of table 12. In other embodiments of aspects of the invention, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NOS: 40-55. In other embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NOS.47-49. In some embodiments, the Q tag comprises LLQGG (SEQ ID NO: 172), GGGLLQGG (SEQ ID NO: 173), RPQGF (SEQ ID NO: 47), or RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Q tag comprises peptide sequence RPQGF (SEQ ID NO: 47). In certain embodiments, the Q tag is selected from the group consisting of RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48), and RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Q tag comprises peptide sequence RPQGFGPP (SEQ ID NO: 49).
In some embodiments, the Q tag is attached to the heavy chain of a SIRP-a antibody. In some embodiments, the Q tag is attached to the heavy chain of the SIRP-a antibody via a linker (e.g., an amino acid or other chemical linker). In some embodiments, the Q tag is attached to the heavy chain (e.g., fused in-frame with the heavy chain) of the SIRP-a antibody. In some embodiments, the Q tag is attached to the C-terminus of the heavy chain of the SIRP-a antibody. In some embodiments, the Q tag is fused to the C-terminus of the heavy chain of the SIRP-a antibody (e.g., fused in frame and linked to the amino acid sequence of the C-terminus). In some embodiments, the Q tag is within the Fc domain of a SIRP-a antibody. In some embodiments, the Q tag is naturally occurring, e.g., present in an antibody (such as in an Fc domain/region) without the need to add another peptide sequence to the antibody. For example, a mutation of N297 to N297A exposes Q295 of the antibody, where binding may occur. In certain embodiments wherein the Fc region comprises a N297A substitution, the conjugate further comprises an immunomodulatory oligonucleotide P linked to a Q295 residue, as shown in the following formula: Wherein L is a linker moiety linked to Q295 via an amide bond.
In some embodiments, the Q tag comprises one or more of the sequences shown in table 12.
TABLE 12Q tag peptide sequences
SEQ ID NO. Peptide sequences
39 LSLSPGLLQGG-OH
40 WPAQGPT
41 WPQGPT
42 WAPQGPT
43 WAQGPT
44 TPGQAPW
45 PNPQLPF
46 RPQQF
47 RPQGF
48 RPQGFPP
49 RPQGFGPP
50 RPRPQQF
51 LSQSKVLG
52 WGGQLL
53 WALQRPHYSYPD
54 WALQRPYTLTES
55 WALQGPYTLTES
In some embodiments, the conjugates provided herein are directed against target-specific cells and tissues in vivo for targeted delivery of conjugated payload oligonucleotides. In certain embodiments, the cell targeted by the conjugates provided herein is a bone marrow cell. In certain embodiments, the cell targeted by the conjugate provided herein is a T cell. In certain embodiments, the cell targeted by the conjugates provided herein is a neutrophil. In certain embodiments, the cells targeted by the conjugates provided herein are monocytes. In certain embodiments, the cell targeted by the conjugate provided herein is a macrophage. In certain embodiments, the cell targeted by the conjugates provided herein is a Dendritic Cell (DC). In certain embodiments, the cell targeted by the conjugate provided herein is a mast cell. In certain embodiments, the cell targeted by the conjugate provided herein is a Tumor Associated Macrophage (TAM). In certain embodiments, the cell targeted by the conjugate provided herein is a Myeloid Derived Suppressor Cell (MDSC).
In some embodiments, the antibodies or conjugates of the present disclosure may be delivered in the form of naked protein-drug conjugates, or in the form of protein-drug conjugates formulated with a carrier, and for example in encapsulated form or as part of a nanocarrier, nanoparticle, liposome, polymeric vesicle, or viral envelope. In some embodiments, the antibodies or conjugates of the disclosure can be delivered intracellularly, for example, by conjugation to a protein transduction domain or mimetic. In some embodiments, the antibodies or conjugates of the present disclosure can be delivered by electroporation or microinjection.
In some embodiments, the conjugates of the present disclosure target more than one cell population or cell type, e.g., from the cell populations or cell types described above. In some embodiments, the conjugates of the present disclosure target monocytes and/or DCs. In some embodiments, the conjugates of the present disclosure target monocytes, neutrophils and DCs. In some embodiments, the conjugates of the present disclosure target monocytes, macrophages, neutrophils and DCs.
In some embodiments, the SIRP-a antibody or conjugate has one or more effector functions, including, but not limited to ADCC and/or ADCP. In some embodiments, the SIRP-a antibody or conjugate comprises a human Fc region, e.g., a human IgG Fc region.
In some embodiments, a SIRP-a antibody or conjugate of the disclosure comprises an antibody constant domain. In some embodiments, a SIRP-a antibody or conjugate of the disclosure comprises an antibody heavy chain constant domain and/or an antibody light chain constant domain listed in table 13. In some embodiments, a SIRP-alpha antibody or conjugate of the present disclosure comprises an antibody heavy chain constant domain selected from the group consisting of SEQ ID NOS 92-107 and 178. In some embodiments, a SIRP-alpha antibody of the present disclosure comprises an antibody heavy chain constant domain having a Q tag at the C-terminus of the Fc region, for example as shown in SEQ ID NO 95 or 178. In some embodiments, a SIRP-a antibody or conjugate of the disclosure comprises two antibody heavy chains each having a constant domain, wherein each of the two antibody heavy chains comprises a Q tag at the C-terminus of the Fc region, e.g., as shown in SEQ ID No. 95 or 178. In some embodiments, a SIRP-a antibody or conjugate of the disclosure comprises two antibody heavy chains each having a constant domain, wherein only one of the two antibody heavy chains comprises a Q tag at the C-terminus of the Fc region, e.g., as shown in SEQ ID NOs 95 or 178. In some embodiments, a SIRP-alpha antibody or conjugate of the present disclosure comprises an antibody light chain constant domain selected from the group consisting of SEQ ID NOS 108-110.
TABLE 13 constant domain sequences of antibodies
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In another aspect of the present disclosure, provided herein is a conjugate comprising a SIRP-alpha antibody or antigen binding fragment thereof and one or more immunomodulatory oligonucleotides (P), wherein the SIRP-alpha antibody or antigen binding fragment is linked to one or more Q-tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47), wherein each immunomodulatory oligonucleotide is linked to the Q-tag peptide via an amide bond to the glutamine residue of the Q-tag peptide and a linker (L), as shown in formula (A),
Wherein:
each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating the point of attachment to the remainder of the conjugate linked to Q via an amide linkage to a glutamine residue;
and each P is independently an immunomodulatory oligonucleotide having the structure:
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection to L;
Wherein Ab comprises a heavy chain Variable (VH) domain and a light chain Variable (VL) domain, wherein the VH domain comprises a CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), a CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and a CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58);
Wherein the VL domain comprises a CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), a CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and a CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61).
In other aspects, provided herein is a conjugate comprising a protein, at least one Q tag peptide sequence comprising a glutamine residue, and at least one immunomodulatory oligonucleotide, wherein the Q tag peptide sequence is naturally occurring or synthetic, and wherein the immunomodulatory oligonucleotide is linked to a Q tag via an amide bond with the glutamine residue, wherein at least one Q tag peptide sequence is selected from the group consisting of SEQ ID NOs 39-55.
In some embodiments, the immunomodulatory oligonucleotide has a sequence selected from the group consisting of the oligonucleotides of table 15 and table 17.
In one aspect, provided herein is a SIRP-a antibody comprising at least one Q tag peptide sequence comprising a glutamine residue. In some embodiments, the Q tag peptide sequence is naturally occurring or synthetic. In certain embodiments, the Q tag peptide sequence is an internal reactive glutamine exposed by an amino acid substitution. In other embodiments, the Q tag is fused to the C-terminus of the heavy chain of the protein. In other embodiments, at least one of the at least one Q tag peptide sequences is selected from the group consisting of SEQ ID NOS: 39-55.
In another aspect of the present disclosure, provided herein is a SIRP-a antibody of formula (B):
wherein:
Each Q is independently a Q tag peptide comprising a peptide sequence having at least one glutamine residue;
ab is an SIRP-alpha antibody or antigen binding fragment thereof; and
E is an integer from 1 to 20.
The SIRP-a antibody of formula (B) may be a precursor to an antibody-oligonucleotide conjugate of formula (a) as described above. Thus, the properties and embodiments of the antibodies as described in the previous aspect of formula (a) may be the same as or different from the properties and/or embodiments of the antibodies of formula (B).
In some embodiments of aspects of the invention, the SIRP-a antibody or fragment thereof is a monoclonal antibody or fragment thereof. In certain embodiments, the SIRP-a antibody or fragment thereof is a Fab, F (ab ') 2, fab' -SH, fv, scFv, single domain, single heavy chain, or single light chain antibody or antibody fragment. In other embodiments, the SIRP-a antibody or fragment thereof is a humanized, human or chimeric antibody or fragment thereof.
In some embodiments, the SIRP-a antibody comprises an Fc region. In certain embodiments wherein the SIRP-a antibody comprises an Fc region, the Fc region is a human Fc region selected from the group consisting of: an IgG1 Fc region, an IgG2 Fc region, and an IgG4 Fc region.
In certain embodiments of aspects of the invention, the Fc region is:
(a) A human IgG1 Fc region comprising an L234A, L a and/or G237A substitution, the amino acid positions being numbered according to the EU index;
(b) A human IgG2 Fc region comprising a330S and/or P331S substitution, amino acid positions numbered according to the EU index; or (b)
(C) Comprising a S228P and/or L235E substituted human IgG4 Fc region, amino acid positions numbered according to the EU index.
In some embodiments, the Fc region further comprises an N297A substitution, the amino acid positions being numbered according to the EU index. In other embodiments, the Fc region further comprises a D265A substitution, the amino acid positions being numbered according to the EU index. In other embodiments, the SIRP-a antibody comprises a human lambda light chain. In other embodiments, the SIRP-a antibody comprises a human kappa light chain.
In some embodiments, at least one Q tag is attached to the heavy chain of a SIRP-a antibody. In certain embodiments, at least one Q tag is fused to the C-terminus of the heavy chain of a SIRP-a antibody. In other embodiments, at least one Q tag is attached to the light chain of a SIRP-a antibody. In other embodiments, at least one Q tag is within the Fc domain.
In some embodiments of aspects of the invention, the SIRP-a antibody is linked to 1 to 20Q tags Q. In certain embodiments, the number of Q tags attached to the SIRP-a antibody conjugate is an integer of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20. In certain other embodiments, 1 or 2Q tags are attached to a SIRP-a antibody or antigen binding fragment. In other embodiments, the number of Q tags attached to the SIRP-a antibody conjugate is an integer from 1 to 10, 10 to 20, 5 to 10, 10 to 15, 15 to 20, or 1 to 5.
In other embodiments of aspects of the invention that may be combined with any of the preceding embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NOs 39-55. In some embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of the peptide sequences of table 12. In other embodiments of aspects of the invention, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NOS: 40-55. In other embodiments, each Q tag independently comprises or is a peptide sequence selected from the group consisting of SEQ ID NOS.47-49. In some embodiments, the Q tag comprises LLQGG (SEQ ID NO: 172), GGGLLQGG (SEQ ID NO: 173), RPQGF (SEQ ID NO: 47), or RPQGFGPP (SEQ ID NO: 49). In some embodiments of aspects of the invention, each Q is independently a Q tag comprising peptide sequence RPQGF (SEQ ID NO: 47). In certain embodiments, each Q tag comprising peptide sequence RPQGF (SEQ ID NO: 47) is selected from the group consisting of RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48), and RPQGFGPP (SEQ ID NO: 49). In certain embodiments, each Q tag independently comprises or is a peptide sequence RPQGFGPP (SEQ ID NO: 49). In some embodiments, the antibody or conjugate comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 87, 88 or 89 and a Q tag of the present disclosure.
Immunomodulatory oligonucleotides
In yet another aspect, provided herein is an immunomodulatory oligonucleotide of formula (C),
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
U 5' is-H or hydrogen;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, optionally wherein at least one of R g1、Rg2、Rg3 and R g4 is oxo, and wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the oxo-linked carbon forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy or 2-methoxyethoxy;
R1-(CH2)3-OH；
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments, if one of R g1、Rg2、Rg3 and R g4 is oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position.
In some embodiments of aspects of the invention, U 5' is-H. In other embodiments, U 5' is halogen. In certain embodiments, U 5' is iodine or bromine. In some embodiments of aspects of the invention, the immunomodulatory oligonucleotide of formula (C) is an immunomodulatory oligonucleotide of formula (C'). In other embodiments of aspects of the invention, the immunomodulatory oligonucleotide of formula (C) is an immunomodulatory oligonucleotide of formula (C ").
In some embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (C):
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, optionally wherein at least one of R g1、Rg2、Rg3 and R g4 is oxo and wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the oxo-linked carbon forms a single bond with the ring nitrogen at the 7 position;
r 3' is methoxy or 2-methoxyethoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments, if one of R g1、Rg2、Rg3 and R g4 is oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position.
In other embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (C "):
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, optionally wherein at least one of R g1、Rg2、Rg3 and R g4 is oxo and wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the oxo-linked carbon forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy or 2-methoxyethoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments, if one of R g1、Rg2、Rg3 and R g4 is oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position.
In some embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (C):
wherein:
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In other embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (C "):
wherein:
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments of aspects of the invention, Z is S. In additional embodiments, the oligonucleotide comprises at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S -. In certain embodiments, the oligonucleotide comprises at least two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S -. The pair of geminal T 1 and T 2, wherein T 1 is S and T 2 is S -, may also be described as phosphorodithioate linkages.
It will be appreciated that in some cases where the oligonucleotide has at least a pair of geminal T 1 and T 2, where T 1 is S and T 2 is S -, one or more phosphorodithioate linkages may be further described in terms of where the linkages are located within the oligonucleotide. The position of the linkage may be characterized, for example, between two nucleoside residues, such as between the first and second nucleoside residues (or between nucleoside residues 1 and 2) counted from the 5' end of the oligonucleotide. Alternatively, the linkage position may be described as being located at the 3' position of a given nucleoside residue, for example at the 3' position of an internucleoside linker or 3' terminal residue immediately following the specified nucleoside residue.
In some embodiments in which the oligonucleotide comprises at least one pair of gem T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 0, at least one phosphorodithioate linkage is between nucleoside residues 1 and 2, between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, between nucleoside residues 10 and 11, or between nucleoside residues 11 and 12. In some embodiments in which the oligonucleotide comprises at least one pair of gem T 1 and T 2 (where T 1 is S and T 2 is S-) and where n is 0, the at least one phosphorodithioate linkage is located at the 3' position of nucleoside residue 1, nucleoside residue 2, nucleoside residue 3, nucleoside residue 5, nucleoside residue 6, nucleoside residue 7, nucleoside residue 8, nucleoside residue 9, nucleoside residue 10, nucleoside residue 11, nucleoside residue 12, or nucleoside residue 13.
In some embodiments in which the oligonucleotide comprises at least one pair of geminal T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 1, at least one phosphorodithioate linkage is between nucleoside residues 1 and 2, between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, between nucleoside residues 10 and 11, between nucleoside residues 11 and 12, or between nucleoside residues 12 and 13. In some embodiments in which the oligonucleotide comprises at least one pair of gem T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 0, the at least one phosphorodithioate linkage is located at the 3' position of nucleoside residue 1, nucleoside residue 2, nucleoside residue 3, nucleoside residue 5, nucleoside residue 6, nucleoside residue 7, nucleoside residue 8, nucleoside residue 9, nucleoside residue 10, nucleoside residue 11, nucleoside residue 12, nucleoside residue 13, or nucleoside residue 14.
In some embodiments in which the oligonucleotide comprises at least one pair of geminal T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 1, at least one phosphorodithioate linkage is between nucleoside residues 1 and 2, between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, between nucleoside residues 10 and 11, between nucleoside residues 11 and 12, or between nucleoside residues 12 and 13. In some embodiments in which the oligonucleotide comprises at least one pair of gem T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 1, the at least one phosphorodithioate linkage is located at the 3' position of nucleoside residue 1, nucleoside residue 2, nucleoside residue 3, nucleoside residue 5, nucleoside residue 6, nucleoside residue 7, nucleoside residue 8, nucleoside residue 9, nucleoside residue 10, nucleoside residue 11, nucleoside residue 12, nucleoside residue 13, or nucleoside residue 14.
In some embodiments in which the oligonucleotide comprises at least one pair of geminal T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 2, at least one phosphorodithioate linkage is between nucleoside residues 1 and 2, between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, between nucleoside residues 10 and 11, between nucleoside residues 11 and 12, between nucleoside residues 12 and 13, or between nucleoside residues 13 and 14. In some embodiments in which the oligonucleotide comprises at least one pair of gem T 1 and T 2 (where T 1 is S and T 2 is S -) and where n is 2, the at least one phosphorodithioate linkage is located at the 3' position of nucleoside residue 1, nucleoside residue 2, nucleoside residue 3, nucleoside residue 5, nucleoside residue 6, nucleoside residue 7, nucleoside residue 8, nucleoside residue 9, nucleoside residue 10, nucleoside residue 11, nucleoside residue 12, nucleoside residue 13, nucleoside residue 14, or nucleoside residue 15.
In other embodiments in which the oligonucleotide has at least two phosphorodithioate linkages or comprises at least two pairs of gems T 1 and T 2 (where T 1 is S and T 2 is S -), the position of one or both phosphorodithioate linkages or pairs of T 1 and T 2 may be specified. It should be appreciated that the position of one or both dithiophosphate linkages may be independently varied.
In some embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (C):
/>
wherein:
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In other embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (C "):
/>
wherein:
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
R 5' is-H or methoxy; r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments of aspects of the invention, Z is S. In additional embodiments, the oligonucleotide comprises at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S -. In certain embodiments, the oligonucleotide comprises at least two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S -.
In yet another embodiment of aspects of the invention, provided herein is an oligonucleotide of formula (C):
/>
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
Provided that the oligonucleotide comprises at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S,
T 3 is a groupWherein/>Indicating the point of attachment to the rest of the oligonucleotide;
Z is O or S;
U 5' is-H or halogen;
r 5' is-H;
R c1 is-H;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-methyl; and
N is 1, and the number of the n is 1,
Or a pharmaceutically acceptable salt thereof.
In some embodiments of any of the foregoing, at least one pair of geminal T 1 and T 2, wherein T 1 is S and T 2 is S, is between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11. In other embodiments of the foregoing, the oligonucleotide comprises at least two pairs of gem T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein the at least two pairs of gem T 1 and T 2 (wherein T 1 is S and T 2 is S) are between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11.
In some embodiments, the oligonucleotide comprises one or two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein the one or two pairs of gems T 1 and T 2 are between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11. In certain embodiments, the oligonucleotide comprises a pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein the pair of gem T 1 and T 2 is located between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11. In certain other embodiments, the oligonucleotide comprises two pairs of gem T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein two pairs of gem T 1 and T 2 (wherein T 1 is S and T 2 is S) are between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11.
In some embodiments, R 5' is H. In other embodiments, R 5' is methoxy. In some embodiments, R c1 is H. In other embodiments, R c1 is methoxy. In other embodiments, R 2 is methyl. In other embodiments, R 2 is H. In other additional embodiments that may be combined with any of the preceding embodiments, T 3 isIn other embodiments, T 3 is. /(I)In certain embodiments, m is an integer from 20 to 25.
In another aspect, the immunomodulatory oligonucleotide of formula (C) is an oligonucleotide selected from the group consisting of the oligonucleotides of table 14 and table 15, or a pharmaceutically acceptable salt thereof. In other embodiments, the immunomodulatory oligonucleotide of formula (C) is an oligonucleotide selected from the group consisting of the oligonucleotides of table 15, or a pharmaceutically acceptable salt thereof.
TABLE 14 modified oligonucleotide Structure (with PEG 3NH2)
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TABLE 15 modified oligonucleotide Structure (with-PEG 3NH2)
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/>
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In some embodiments, the immunomodulatory oligonucleotides of formula (C) may be used as precursors to prepare conjugates comprising a SIRP-a antibody or antigen binding fragment thereof linked via a Q tag and one or more immunomodulatory oligonucleotides of formula (C), as shown in the structures of formula (a) described herein.
In some embodiments, the immunomodulatory oligonucleotide of formula (C) may be modified to attach a linker moiety L to a terminal group T 3 in formula (C) to provide the immunomodulatory oligonucleotide of formula (D). In yet another aspect, provided herein is an immunomodulatory oligonucleotide of formula (D):
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
L is a group Wherein m is an integer from 0 to 50 and wherein/>Indicating the point of attachment to the rest of the oligonucleotide via T 3;
Z is O or S;
U 5' is-H or halogen;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, optionally wherein at least one of R g1、Rg2、Rg3 and R g4 is oxo and wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the oxo-linked carbon forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy or 2-methoxyethoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments, if one of R g1、Rg2、Rg3 and R g4 is oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position.
In some embodiments of aspects of the invention, U 5' is-H. In other embodiments, U 5' is halogen. In certain embodiments, U 5' is iodine or bromine. In some embodiments of aspects of the invention, the immunomodulatory oligonucleotide of formula (D) is an immunomodulatory oligonucleotide of formula (D'). In other embodiments of aspects of the invention, the immunomodulatory oligonucleotide of formula (D) is an immunomodulatory oligonucleotide of formula (D ").
In some embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (D'):
/>
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
L is a group Wherein m is an integer from 0 to 50 and wherein/>Indicating the point of attachment to the rest of the oligonucleotide via T 3;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, optionally wherein at least one of R g1、Rg2、Rg3 and R g4 is oxo and wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the oxo-linked carbon forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy or 2-methoxyethoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments, if one of R g1、Rg2、Rg3 and R g4 is oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position.
In other embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (D "):
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
L is a group Wherein m is an integer from 0 to 50 and wherein/>Indicating the point of attachment to the rest of the oligonucleotide via T 3;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H or oxo, optionally wherein at least one of R g1、Rg2、Rg3 and R g4 is oxo and wherein if one R g1、Rg2、Rg3 and R g4 are oxo, the oxo-linked carbon forms a single bond with the ring nitrogen at the 7 position;
R 3' is methoxy or 2-methoxyethoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments, if one of R g1、Rg2、Rg3 and R g4 is oxo, the carbon to which the oxo is attached forms a single bond with the ring nitrogen at the 7 position.
In some embodiments of aspects of the invention, the invention also provides an immunomodulatory oligonucleotide of formula (D'):
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
L is a group Wherein m is an integer from 0 to 50 and wherein/>Indicating the point of attachment to the rest of the oligonucleotide via T 3;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In other embodiments of aspects of the invention, provided herein is an immunomodulatory oligonucleotide of formula (D "):
Wherein the method comprises the steps of
Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
L is a group Wherein m is an integer from 0 to 50 and wherein/>Indicating the point of attachment to the rest of the oligonucleotide via T 3;
Z is O or S;
R 5' is-H or methoxy;
r c1 is-H or methoxy;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer of 0 to 2,
Or a pharmaceutically acceptable salt thereof.
In some embodiments of aspects of the invention, Z is S. In additional embodiments, the oligonucleotide comprises at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S -. In certain embodiments, the oligonucleotide comprises at least two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S -.
In yet another embodiment of aspects of the invention, provided herein is an oligonucleotide of formula (D):
Wherein the method comprises the steps of Indicating the point of attachment within the oligonucleotide;
each T 1 is independently O or S;
Each T 2 is S -;
Provided that the oligonucleotide comprises at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S,
T 3 is a groupWherein/>Indicates the point of attachment to L, and wherein/>Indicating the point of attachment to the rest of the oligonucleotide;
L is a group Wherein m is an integer from 0 to 50 and wherein/>Indicating the point of attachment to the rest of the oligonucleotide via T 3;
Z is O or S;
U 5' is-H or halogen;
r 5' is-H;
R c1 is-H;
R g1、Rg2、Rg3 and R g4 are H;
R 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is methyl; and
N is 1, and the number of the n is 1,
Or a pharmaceutically acceptable salt thereof.
In some embodiments of any of the foregoing, at least one pair of geminal T 1 and T 2, wherein T 1 is S and T 2 is S, is between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11. In other embodiments of the foregoing, the oligonucleotide comprises at least two pairs of gem T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein the at least two pairs of gem T 1 and T 2 (wherein T 1 is S and T 2 is S) are between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11.
In some embodiments, the oligonucleotide comprises one or two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein the one or two pairs of gems T 1 and T 2 are between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11. In certain embodiments, the oligonucleotide comprises a pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein the pair of gem T 1 and T 2 is located between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11. In certain other embodiments, the oligonucleotide comprises two pairs of gem T 1 and T 2, wherein T 1 is S and T 2 is S, and wherein two pairs of gem T 1 and T 2 (wherein T 1 is S and T 2 is S) are between nucleoside residues 2 and 3, between nucleoside residues 3 and 4, between nucleoside residues 5 and 6, between nucleoside residues 6 and 7, between nucleoside residues 7 and 8, between nucleoside residues 8 and 9, between nucleoside residues 9 and 10, or between nucleoside residues 10 and 11.
In some embodiments, R 5' is H. In other embodiments, R 5' is methoxy. In some embodiments, R c1 is H. In other embodiments, R c1 is methoxy. In other embodiments, R 2 is methyl. In other embodiments, R 2 is H. In other additional embodiments that may be combined with any of the preceding embodiments, T 3 isIn other embodiments, T 3 is/>In certain embodiments, m is an integer from 20 to 25.
In another aspect, the immunomodulatory oligonucleotide of formula (D) is an oligonucleotide selected from the group consisting of the oligonucleotides of table 16 and table 17, or a pharmaceutically acceptable salt thereof. In other embodiments of this aspect, the oligonucleotide of formula (D) is selected from the group consisting of the oligonucleotides of table 17 or a pharmaceutically acceptable salt thereof.
TABLE 16 modified oligonucleotide Structure (with-PEG 3NHCOPEG24NH2)
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TABLE 17 modified oligonucleotide Structure (with-PEG 3NHCOPEG24NH2)
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Like the oligonucleotides of formula (C), the immunomodulatory oligonucleotides of formula (D) can be used as precursors to prepare a kit comprising a SIRP-a antibody or antigen binding fragment thereof linked via a Q tag and one or more immunomodulatory oligonucleotides of formula (D), as shown in the structures of formula (a) described herein.
Immunomodulatory oligonucleotides of formulae (C) and (D) as described herein can be prepared according to methods known in the art. General methods for preparing immunomodulatory oligonucleotides are described below, including those provided in the present disclosure.
General oligonucleotide synthesis:
general procedure:
Experimental details:
automated oligonucleotide synthesis (1. Mu. Mol scale) was performed on MerMade or 12 with the following reagents and solvents:
oxidant-0.02M THF/pyridine/H 2 O with I 2 (60 second oxidation per cycle),
Sulfiding reagent II-dithiazole derivative/pyridine/acetonitrile (0.05M in 6:4 pyridine: acetonitrile) (60 seconds per cycle)
Deblock-3% trichloroacetic acid (2X 40 sec per cycle deblock),
End-capping mixture A-THF/2, 6-lutidine/Ac 2 O (end capping 60 seconds per cycle), and
Blocking mixture B-16% methylimidazole-containing THF (60 seconds per cycle blocking)
The exception of standard oligonucleotide synthesis conditions is as follows:
CPG vector with non-nucleoside linker called Uny linker was used.
Prior to synthesis, all 2 '-deoxyribose-phosphoramidites were resuspended to 100mM in 100% anhydrous acetonitrile, depending on the solubility of the starting material, except that some of the modified 2' -deoxy-phosphoramidite was dissolved to 100mM in THF/acetonitrile mixture (1:4).
Phosphoramidite activation is performed with a 2.5-fold molar excess of 5-benzylthio-1H-tetrazole (BTT). The activated 2' -deoxyribose-phosphoramidite was coupled at 2 x 1min per insertion and the modified phosphoramidite was coupled at 2 x 3 min per insertion.
Sulfiding the backbone with 0.05M pyridine/acetonitrile containing sulfiding agent II (6:4) for 1min.
Oligonucleotide deprotection and purification protocol:
After automated oligonucleotide synthesis, the solid support and base protecting groups (such as A-Bz, C-Ac, G-iBu, etc.) and methyl esters of phosphotriesters are cleaved and deprotected in 1mL AMA (1:1 ratio of 36% aqueous ammonia and 40% methylamine/methanol) at room temperature for 2 hours or more, followed by centrifugation.
The crude oligonucleotide pellet (pellet) was resuspended in 100 μl of 50% acetonitrile, briefly heated to 65 ℃ and vortexed well.
For oligonucleotide purification, 100 μl of crude oligonucleotide was injected onto RP-HPLC with the following buffer/gradient:
buffer a=50 mM TEAA/water;
Buffer b=90% acetonitrile; and
-Flow rate = 1mL/min;
-gradient:
0-2min (100% buffer A/0% buffer B),
2-42Min (0% to 60% buffer B), and
42-55Min (60% to 100% buffer B).
DBCO conjugation and purification scheme:
DBCO NHS esters were conjugated with crude 2' -deoxy DMT-oligonucleotides as described herein. The crude oligonucleotide pellet was suspended in 45 μl DMSO, briefly heated to 65 ℃ and vortexed well. mu.L of DIPEA was added followed by DBCO-NHS ester (30 eq.) pre-dissolved in DMSO (1M). The reaction was allowed to stand for 10 minutes or until product formation was confirmed by MALDI. A total of 80. Mu.L of crude oligonucleotide sample was injected onto RP-HPLC with the following buffer/gradient:
buffer a=50 mM TEAA/water
Buffer b=90% acetonitrile
-Flow rate = 1mL/min
-Gradient:
o0-2 min (90% buffer A/10% buffer B)
2-42Min (0% to 60% buffer B)
42-55Min (60% to 100% buffer B).
Across the major RP-HPLC peak, 0.5mL fractions were collected and analyzed by MALDI-TOF mass spectrometry to confirm the presence of the desired mass. Purified fractions of selected mass were frozen and lyophilized. Once dried, the fractions were resuspended, combined with the corresponding fractions, frozen and lyophilized.
DMT cleavage: the lyophilized pellet was suspended in 20 μl of 50% acetonitrile and 80 μl of acetic acid was added, and the sample was left to stand at room temperature for 1 hour, frozen and lyophilized. The dried sample was redissolved in 20% acetonitrile and desalted via a NAP 10 (Sephadex TM -G25 DNA grade) column. For the final product, the collected pure fractions were frozen and lyophilized.
Method for ligating oligonucleotides to a ligation moiety
Cu catalyzed click reaction
Copper-THPTA complex preparation
An aqueous solution of 5mM copper sulfate pentahydrate (CuSO 4-5H2 O) and an aqueous solution of 10mM tris (3-hydroxypropyl triazolylmethyl) amine (THPTA) were mixed at 1:1 (v/v) (1:2 molar ratio) and allowed to stand at room temperature for 1 hour. This complex can be used to catalyze Hu Yisi cycloadditions (Huisgen cycloaddition), for example as shown in the general conjugation scheme below.
General procedure (100 nM scale):
To a solution of 710. Mu.L of water and 100. Mu.L of t-butanol (10% final volume) in a 1.7mL Eppendorf tube was added 60. Mu.L of copper-THPTA complex followed by 50. Mu.L of 2mM oligo solution, 60. Mu.L of 20mM sodium ascorbate aqueous solution and 20. Mu.L of 10mM targeting moiety-azide solution. After thorough mixing, the solution was allowed to stand at room temperature for 1 hour. The completion of the reaction was confirmed by gel analysis. The reaction mixture is added to a reaction mixture containing 5 to 10 times molar excess TAAcona (resin-bonded sodium EDTA salt) in a screw cap vial. The mixture was stirred for 1 hour. Next, the mixture was eluted through illustra TMNapTM -10 column Sephadex TM. The resulting solution was then frozen and lyophilized overnight.
Ligation via amide linkage:
Conjugation via amidation may be performed under amidation reaction conditions known in the art. See, for example, aaronson et al Bioconjugate chem.22:1723-1728,2011.
Wherein the method comprises the steps of
Each q is 0 or 1;
each m is an integer from 0 to 5;
Z is O or S;
R O is a bond to a nucleoside in an oligonucleotide;
R is a bond to H, a nucleoside in an oligonucleotide, a solid support or a capping group (e.g., - (CH 2)3 -OH);
Each R' is independently H, -Q 1-QA1, a bioreversible group or a non-bioreversible group;
Each R' is independently H, -Q 1-QA-Q1 -T, a bioreversible group or a non-bioreversible group; each R A is independently H OR-OR C, wherein R C is-Q 1-QA1, a bioreversible group, OR an abiotic reversible group;
Each R B is independently H OR-OR D, wherein R D is-Q 1-QA-Q2 -T, a bioreversible group, OR an abiotic reversible group;
Wherein the method comprises the steps of
Each Q 1 is independently a divalent, trivalent, tetravalent, or pentavalent group, one of which is bound to Q A or Q A1, the second valency is open, and each of the remaining valencies (if present) is independently bound to a secondary moiety;
Each Q 2 is independently a divalent, trivalent, tetravalent, or pentavalent group, with one valence being bound to Q A, a second valence being bound to T, and each of the remaining valences (if present) being independently bound to a secondary moiety;
Q A is a compound containing-C (O) -N (H) -or-N (H) -optionally substituted C 2-12 heteroalkylene with C (O) -;
Q A1 is-NHR N1 or-COOR 12 wherein R N1 is a H, N-protecting group or an optionally substituted C 1-6 alkyl group and R 12 is H, an optionally substituted C 1-6 alkyl group or an O-protecting group; and
T is a connecting portion of the metal-insulator-metal composite,
Provided that the starting material contains at least one-Q 1-QA1 and the product contains-Q 1-QA-Q2 -T.
Solution phase connection:
Wherein the method comprises the steps of
M is an integer from 0 to 5;
Z is O or S;
R O is a bond to a nucleoside in an oligonucleotide;
R is a bond to H, a nucleoside or a capping group in the oligonucleotide;
each R' is independently H, -Q 1-NH2, a bioreversible group or a non-bioreversible group;
Each R' is independently H, -Q 1-NH-CO-Q2 -T, a bioreversible group or a non-bioreversible group;
Each R A is independently H OR-OR C, wherein R C is-Q 1-NH2, a bioreversible group, OR an abiotic reversible group;
Each R B is independently H OR-OR D, wherein R D is-Q 1-NH-CO-Q2 -T, a bioreversible group, OR an abiotic reversible group;
Wherein the method comprises the steps of
Each Q 1 is independently a divalent, trivalent, tetravalent, or pentavalent group, one valence being bound to-NH-CO-or-NH 2, the second valence being open, and each of the remaining valences (if present) being independently bound to a secondary moiety;
Each Q 2 is independently a divalent, trivalent, tetravalent, or pentavalent group, one of the valences being bound to-NH-CO-, the second valency being a bond to T, and each of the remaining valences (if present) being independently bound to an auxiliary moiety; and
T is a connecting portion of the metal-insulator-metal composite,
Provided that the starting material contains-Q 1-NH2 and the product contains-Q 1-NH-CO-Q2 -T.
And (3) connection on a carrier:
Wherein the method comprises the steps of
Z is O or S;
R O is a bond to a nucleoside in an oligonucleotide;
Each Q 2 is independently a divalent, trivalent, tetravalent, or pentavalent group, one of the valences being bound to-NH-CO-, the second valency being a bond to T, and each of the remaining valences (if present) being independently bound to an auxiliary moiety; and
T is a linking moiety.
Wherein the method comprises the steps of
N is an integer from 1 to 8;
a is O or-CH 2 -;
Z is O or S;
R O is a bond to a nucleoside in an oligonucleotide;
Each Q 2 is independently a divalent, trivalent, tetravalent, or pentavalent group; wherein one valence is bound to azide or triazole, a second valence is bound to T, and each of the remaining valences (if present) is independently bound to a secondary moiety; and
T is a linking moiety.
Wherein the method comprises the steps of
N is an integer from 1 to 8;
a is O or-CH 2 -;
Z is O or S;
R O is a bond to a nucleoside in an oligonucleotide;
Each Q 2 is independently a divalent, trivalent, tetravalent, or pentavalent group; wherein one valence is bound to azide or triazole, a second valence is bound to T, and each of the remaining valences (if present) is independently bound to a secondary moiety; and
T is a linking moiety.
Wherein the method comprises the steps of
N is an integer from 1 to 8;
a is O or-CH 2 -;
Z is O or S;
R O is a bond to a nucleoside in an oligonucleotide;
Each Q 2 is independently a divalent, trivalent, tetravalent, or pentavalent group; wherein one valence is bound to azide or triazole, a second valence is bound to T, and each of the remaining valences (if present) is independently bound to a secondary moiety; and
Each T is independently a linking moiety.
Representative examples of Fmoc deprotection of phosphotriesters:
An oligonucleotide comprising a phosphotriester with an Fmoc-protected amine is subjected to deprotection conditions, resulting in Fmoc deprotection without observable conversion of the phosphotriester to phosphodiester.
TCCATGACGTTCCTGACGTT(SEQ ID NO:176)
DBCO-NHS conjugated to TCCATGACGTTCCTGACGTT (SEQ ID NO: 176) - -representative examples:
Conjugation of DBCO-NHS to the amino groups in the phosphotriester was completed in 10 minutes at room temperature as demonstrated by mass spectrometry analysis.
RP-HPLC purification of TCCATGACGTTCCTGACGTT (SEQ ID NO: 176) containing a DBCO conjugate group was performed using the following conditions:
buffer a=50 mMTEAA/water;
Buffer b=90% acetonitrile; and
-Flow rate = 1mL/min;
-gradient:
0-2min (100% buffer A/0% buffer B),
2-22Min (0% to 100% buffer B), and
Omicron 22-25min (100% buffer B).
Similar procedures can be used to prepare oligonucleotides using, for example, 2' -modified nucleoside phosphoramidites, such as those described herein. Such procedures are provided in international patent application PCT/US 2015/034749; the disclosure of disulfide phosphotriester oligonucleotide synthesis in PCT/US2015/034749 is incorporated herein by reference.
Conjugation process
Provided herein are methods for preparing conjugates comprising a SIRP-a antibody or antigen binding fragment thereof linked via one or more Q tag peptides and one or more immunomodulatory oligonucleotides, as shown in the structure of formula (a). In some embodiments, the method comprises combining a SIRP-a antibody comprising at least one Q tag peptide sequence with at least one exposed glutamine residue and an oligonucleotide under conditions sufficient to induce binding (i.e., amidation reaction between CpG and Q tag). In other embodiments, the method comprises reacting a SIRP-a antibody comprising at least one Q tag peptide sequence with at least one exposed glutamine residue and an oligonucleotide under chemical conditions sufficient to induce binding. In other embodiments, the methods comprise reacting a SIRP-a antibody comprising at least one Q tag peptide sequence with at least one exposed glutamine residue and an oligonucleotide under conditions sufficient to induce conjugation of the enzyme (e.g., with transglutaminase).
Transglutaminase mediated conjugation reaction conditions
In one aspect, provided herein is a method of preparing a conjugate of formula (a), the method comprising combining one or more immunomodulatory oligonucleotides (P) with a SIRP-a antibody comprising one or more glutamine residues. In one aspect, provided herein is a method of making a conjugate comprising a SIRP-alpha antibody or antigen binding fragment (Ab) and one or more immunomodulatory oligonucleotides (P), wherein the SIRP-alpha antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47), and wherein each immunomodulatory oligonucleotide is linked to a Q tag peptide via an amide bond to a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (A),
Wherein:
indicating the point of attachment of each Q to the SIRP-a antibody or antigen binding fragment (Ab) thereof;
each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
each L is independently a bond or a linker moiety linked to Q via an amide bond with a glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide;
the method comprises contacting a compound of formula (B) with one or more immunomodulatory oligonucleotides P
Wherein Ab and Q are as defined above for formula (A) and e is an integer from 1 to 20,
Wherein each P independently has the formula:
Wherein the method comprises the steps of
X 5 'is a 5' terminal nucleoside;
X 3 'is a 3' terminal nucleoside;
y PTE is an internucleoside phosphotriester;
y 3' is a terminal phosphotriester;
Each X N is independently a nucleoside;
each Y N is independently an internucleoside linker;
b and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5; and
L is a linker moiety having a terminal amine,
The contacting occurs in the presence of a transglutaminase.
In another aspect, a method for preparing a conjugate comprising a SIRP-alpha antibody or antigen binding fragment thereof (Ab) and one or more immunomodulatory oligonucleotides (P), wherein the SIRP-alpha antibody or antigen binding fragment is linked to one or more Q-tag peptides (Q) comprising at least one glutamine residue, wherein each immunomodulatory oligonucleotide is linked to a Q-tag peptide via an amide bond to a glutamine residue of the Q-tag peptide and a linker (L), as shown in formula (A),
Wherein:
indicating the point of attachment of each Q to the SIRP-a antibody or antigen binding fragment (Ab) thereof;
Each Q is independently a Q tag peptide having at least one glutamine residue;
each L is independently a bond or a linker moiety linked to Q via an amide bond with a glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide;
The method comprises contacting a compound of formula (B) with one or more immunomodulatory oligonucleotides P in the presence of transglutaminase
Wherein Ab and Q are as defined above for formula (A) and e is an integer from 1 to 20,
Wherein each oligonucleotide P is independently an immunomodulatory oligonucleotide of formula (C) or formula (D).
In some embodiments, the conjugate comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more Q tag peptides. In some embodiments, the conjugate comprises one, two, three, four, five, six, seven, eight, nine, ten, or twenty Q tag peptides. In some embodiments, the conjugate has 2Q tag peptides. In some embodiments, the conjugate comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or twenty or more immunomodulatory oligonucleotides. In some embodiments, the conjugate comprises one, two, three, four, five, six, seven, eight, nine, ten, or twenty immunomodulatory oligonucleotides. In some embodiments, the conjugate has an immunomodulatory oligonucleotide.
In another aspect, the method comprises combining a compound of formula (C) with a SIRP-a antibody of formula (B) comprising one or more glutamine residues in the presence of transglutaminase. In some embodiments, the methods comprise contacting a compound of formula (D) with a SIRP-a antibody of formula (B) comprising one or more glutamine residues in the presence of transglutaminase. In some embodiments, the final concentration of the compound of formula (C) or formula (D) is in the range of about 1-100. Mu.M. In some embodiments, the final concentration of antibody comprising the Q tag is in the range of about 1-500 μm. In some embodiments, the final concentration of transglutaminase is in the range of about 1-500 μm. In some embodiments, the final concentration of transglutaminase is in the following range: about 1-50. Mu.M, about 50-100. Mu.M, about 100-150. Mu.M, about 150-200. Mu.M, about 200-250. Mu.M, about 250-300. Mu.M, about 300-400. Mu.M, about 400-500. Mu.M, about 100-125. Mu.M, about 125-150. Mu.M, about 150-175. Mu.M, about 175-200. Mu.M, about 200-225. Mu.M, about 225-250. Mu.M, about 250-275. Mu.M, about 275-300. Mu.M, about 300-325. Mu.M, or about 325-350. Mu.M.
In some embodiments, the ratio of the antibody comprising the Q tag to the compound of formula (C) or formula (D) is in the following range by weight: about 1:1-250:1, about 1:1-5:1, about 5:1-10:1, about 10:1-20:1, about 20:1-30:1, about 30:1-40:1, about 40:1-50:1, about 50:1-75:1, about 75:1-100:1, about 100:1-150:1, about 150:1-200:1, about 200:1-250:1, about 1:1-25:1, about 25:1-50:1, about 50:1-75:1, about 75:1-100:1, or about 100:1-250:1). In some embodiments, the ratio of compound of formula (C) or formula (D) to antibody is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, or about 20:1 in molar concentration.
In some embodiments, the ratio of antibody comprising a Q tag to transglutaminase is in the following range by weight: about 1:1-500:1, about 1:1-5:1, about 5:1-10:1, about 10:1-20:1, about 20:1-30:1, about 30:1-40:1, about 40:1-50:1, about 50:1-75:1, about 75:1-100:1, about 100:1-150:1, about 150:1-200:1, about 200:1-250:1, about 1:1-25:1, about 25:1-50:1, about 50:1-75:1, about 75:1-100:1, about 100:1-150:1, about 150:1-200:1, about 200:1-250:1, about 250:1-300:1, about 300:1-400:1, or about 400:1-500:1. In some embodiments, the ratio of peptide to transglutaminase is about 15:1, about 16:1, about 17:1, about 18:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, or about 50:1 in molar concentration.
In some embodiments, the ratio of Q-tag to CpG-transglutaminase is about 1:1.3:10. In some embodiments, the ratio of Q-tag to CpG-transglutaminase is about 1:1.5:10. In some embodiments, the ratio of Q-tag to CpG-transglutaminase is about 1:1.3:15.
In some embodiments, the reactants are incubated at greater than 15 ℃, greater than 20 ℃, greater than 25 ℃, greater than 30 ℃, greater than 35 ℃, greater than 40 ℃, greater than 45 ℃, or greater than 50 ℃. In some embodiments, the reactants are incubated at about room temperature. In some embodiments, the reactants are incubated for at least 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 60 hours.
In some embodiments, the methods described herein result in about 5% more, about 10% more, about 15% more, about 20% more, about 25% more, about 30% more, about 35% more, about 40% more, about 45% more, about 50% more, about 60% more, about 70% more, about 80% more, about 90% more, about 95% more, about 97% more, or about 99% more of the compound of formula (a) than the peptide.
In some embodiments, the pH of the reaction is in the range of about 4-10. In some embodiments, the pH of the reaction is in the range of about 4-6, about 6-8, or about 8-10. In some embodiments, the pH of the reaction is in the range of about 7-8.
In another aspect, reactions suitable for ligating a ligating moiety to an oligonucleotide are known in the art, including but not limited to Hu Yisi cycloadditions (Hu isgen cycloaddition) (metal catalyzed or metal free) between an azido group and an alkyne-based conjugation group (e.g., an optionally substituted C 6-16 heterocyclylene group containing an endocyclic carbon-carbon triple bond, or an optionally substituted C 8-16 cycloalkynyl group) to form a triazole moiety; diels-Alder reaction between dienophiles and dienes/heterodienes; bond formation via a cyclic reaction (such as an alkene reaction); amide or thioamide bond formation; sulfonamide bond formation (e.g., with azido compounds); alcohol or phenol alkylation (e.g., williamson alkylation), condensation reactions to form oxime, hydrazone, or amine urea groups; conjugation addition reactions using nucleophiles (e.g., amines and thiols); disulfide bond formation; and nucleophilic substitution at carbonyl (e.g., at an activated carboxylate such as pentafluorophenyl (PFP) ester or Tetrafluorophenyl (TFP) ester) or at electrophilic aromatic (e.g., SNAr at oligofluorinated aromatic, fluorobenzonitrile, or fluoronitrophenyl groups) (e.g., with amine, thiol, or hydroxyl nucleophiles).
In certain embodiments, the linking reaction is a dipolar cycloaddition, and the conjugate moiety comprises an azide group, an optionally substituted C 6-16 heterocyclyl group containing an endocyclic carbon-carbon triple bond, or an optionally substituted C 8-16 cycloalkynyl group. Complementary reactive groups and conjugate groups are selected for their complementarity. For example, an azide is used in one of the conjugate group and the complementary reactive group, and an alkyne is used in the other of the conjugate group and the complementary reactive group.
In some embodiments, each Q tag peptide sequence comprises peptide sequence RPQGFGPP (SEQ ID NO: 49). In some embodiments, the Ab comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides (Q) having at least one glutamine residue; one Q tag peptide is attached to the C-terminus of each of the two antibody heavy chains. In some embodiments, the DAR of the conjugate is 1 or 2. In some embodiments, the DAR of the conjugate is 1, and the method further comprises isolating the conjugate with DAR 1 from the free oligonucleotide, unconjugated antibody, and the conjugate with DAR 2.
Ligation of a linker moiety to an oligonucleotide
The linking moiety may be attached to the oligonucleotide by forming a bond between the linking group in the oligonucleotide and a complementary reactive group bound to the linking moiety. In certain embodiments, the linking moiety is modified to include complementary reactive groups. Methods of introducing such complementary reactive groups into the linking moiety are known in the art.
In certain embodiments, the complementary reactive group is an optionally substituted C 2-12 alkynyl, an optionally substituted N-protected amino, azido, N-maleimido, S-protected thiol,Or an N-protected portion thereof,Optionally substituted C 6-16 heterocyclyl containing an endocyclic carbon-carbon triple bond (e.g./>) 1,2,4, 5-Tetrazine groups (e.g) Optionally substituted C 8-16 cycloalkynyl (e.g) -NHR N1, optionally substituted C 4-8 strained cycloalkenyl (e.g. trans-cyclooctenyl or norbornenyl), or optionally substituted C 1-16 alkyl containing-COOR 12 or-CHO;
wherein:
r N1 is H, N-protecting group or optionally substituted C 1-6 alkyl;
Each R 12 is independently H, optionally substituted C 1-6 alkyl, or an O-protecting group (e.g., a carboxyl protecting group); and
R 13 is halogen (e.g., F).
In certain embodiments, the complementary reactive groups are protected until the conjugation reaction. For example, the protected complementary reactive group may include-COOR PGO or-NHR PGN, where R PGO is an O-protecting group (e.g., a carboxyl protecting group) and R PGN is an N-protecting group.
VI pharmaceutical composition
The SIRP-a antibodies and conjugates of the invention, such as conjugates comprising the structure of formula (a), antibodies of formula (B), and immunomodulatory oligonucleotides of formulae (C), (C '), (C "), (D'), and (D"), or a pharmaceutically acceptable salt of any of the foregoing, or any subgroup thereof, may be formulated into various pharmaceutical forms for administration purposes. As suitable compositions all compositions normally used for systemic administration of drugs can be cited. For the preparation of the pharmaceutical compositions of the present invention, an effective amount of the particular compound (optionally in addition salt form) as the active ingredient is combined in admixture with a pharmaceutically acceptable carrier, which carrier may take many forms depending on the form of preparation desired for administration. These pharmaceutical compositions in unit dosage form are desirable and are particularly suitable for administration orally, rectally, transdermally or by parenteral injection. For example, in preparing the composition in oral dosage form, any of the usual pharmaceutical media may be employed, such as: in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions, water, glycols, oils, alcohols and the like; or in the case of powders, pills, capsules and tablets, solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like. Because of the ease of administration of tablets and capsules, they represent the most advantageous oral unit dosage form, in which case solid pharmaceutical carriers are employed. For parenteral compositions, the carrier will typically comprise sterile water, which is at least a major portion, but may include other ingredients, for example, to aid in dissolution. For example, an injectable solution may be prepared wherein the carrier comprises saline solution, dextrose solution, or a mixture of saline and dextrose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are to be converted to liquid form preparations shortly before use. In compositions suitable for transdermal administration, the carrier optionally comprises a penetration enhancer and/or a suitable humectant, optionally in combination with a minor proportion of any suitable additive of a nature that does not have a significant deleterious effect on the skin. The compounds of the present invention may also be administered via oral inhalation or insufflation in the form of a solution, suspension or dry powder using any delivery system known in the art.
The aforementioned pharmaceutical compositions are particularly advantageous in terms of ease of administration and uniformity of dosage, formulated into unit dosage forms. A unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, powder tablets, injectable solutions or suspensions and the like, and segregated multiples thereof.
Administration may be, but is not limited to, intravenous, intra-arterial, subcutaneous, intraperitoneal, subcutaneous (e.g., via an implanted device), and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.
Pharmaceutical compositions comprising the conjugates described herein may be delivered to cells, cell populations, tumors, tissues, or subjects using delivery techniques known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid-protein conjugate (in vitro or in vivo) may be suitable for use with the compositions described herein. For example, delivery may be achieved by: topical administration (e.g., direct injection, implantation, or topical administration), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.
Thus, in some embodiments, the pharmaceutical compositions described herein may comprise one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein may be formulated for administration to a subject.
As used herein, a pharmaceutical composition or agent includes a pharmacologically effective amount of at least one of the described therapeutic compounds or conjugates and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the active pharmaceutical ingredient (API, therapeutic product) that are intended to be included in the drug delivery system. The excipient does not exert or is not intended to exert a therapeutic effect at the predetermined dosage. Excipients may be used to a) aid in processing the drug delivery system during manufacture, b) protect, support, or enhance stability, bioavailability, or patient acceptance of the API, c) aid in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness of API delivery during storage or use. The pharmaceutically acceptable excipient may or may not be an inert substance.
Excipients include, but are not limited to: absorption enhancers, anti-tackifiers, defoamers, antioxidants, binders, buffers, carriers, coating agents, pigments, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (in the case of water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, cremophor ELTM (BASF, parsippany, N.J.), or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size (in the case of dispersions) and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition absorption delaying agents, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by the following method: the desired amount of active compound is incorporated in the appropriate solvent together with one or a combination of the ingredients listed above, followed by filter sterilization as required. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle which contains an alkaline dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Formulations suitable for intra-articular administration may be in the form of sterile aqueous preparations of the drug, which may be in microcrystalline form, for example in the form of an aqueous microcrystalline suspension. Lipid formulations or biodegradable polymer systems may also be used to allow for intra-articular and ocular administration of drugs.
The active compounds can be prepared with carriers that will prevent rapid elimination of the compound from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The compounds or conjugates can be formulated in unit dosage forms into compositions for ease of administration and dose uniformity. A unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound associated with the desired drug carrier calculated to produce the desired therapeutic effect. The specification of the unit dosage form of the present disclosure is dictated by and directly dependent upon: the unique characteristics of the active compounds and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such active compounds for the treatment of individuals.
The pharmaceutical composition may contain other additional components common in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics or anti-inflammatory agents (e.g., antihistamines, diphenhydramine (DIPHENHYDRAMINE), etc.).
In general, an effective amount of the active compound will be in the range of about 0.1 to about 100mg/kg body weight/day, for example about 1.0 to about 50mg/kg body weight/day. In some embodiments, an effective amount of the active compound will be in the range of about 0.25 to about 5mg/kg body weight/dose. In some embodiments, an effective amount of the active compound will be in the range of 25-400mg every 1 to 18 weeks or 1 to 6 months. In some embodiments, an effective amount of the active compound will be in the range of 50-125mg every 4 weeks or every month. In some embodiments, an effective amount of the active ingredient will be in the range of about 0.5 to about 3mg/kg body weight/dose. In some embodiments, an effective amount of the active ingredient will be in the range of about 25-400 mg/dose. In some embodiments, an effective amount of the active ingredient will be in the range of about 50-125 mg/dose. The amount administered will also likely depend on variables such as the overall health of the patient, the relevant biological efficacy of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, and the route of administration. Furthermore, it will be appreciated that the initial dose administered may be increased beyond the upper level to quickly achieve the desired blood or tissue level, or the initial dose may be less than optimal.
For the treatment of a disease or forming a medicament or composition for the treatment of a disease, a pharmaceutical composition described herein including a SIRP-a antibody or conjugate may be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: second or other conjugates, small molecule drugs, antibodies, antibody fragments, and/or vaccines.
The described SIRP-a antibodies or conjugates can be packaged in a kit, container, package, or dispenser when added to a pharmaceutically acceptable excipient or adjuvant. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.
VII medicine box
Also provided herein is a kit comprising a conjugate as described above.
In another aspect, the kit further comprises a pharmaceutical instruction comprising, but not limited to, appropriate instructions for the preparation and administration of the formulation, side effects of the formulation, and any other relevant information. The instructions may be in any suitable format including, but not limited to, printed matter, video tape, computer readable magnetic disk, optical disk, or instructions for internet-based instructions.
In another aspect, a kit for treating an individual suffering from or susceptible to a disorder described herein is provided, the kit comprising a first container containing an administered amount of a composition or formulation as disclosed herein and instructions for use of the drug. The container may be any of those known in the art and suitable for storing and delivering intravenous formulations. In certain embodiments, the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, or the like for preparing a formulation to be administered to an individual.
In another aspect, kits may also be provided that contain a sufficient dose of a composition described herein (including pharmaceutical compositions thereof) to provide effective treatment to an individual over an extended period of time, such as 1-3 days, 1-5 days, one week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, or longer.
In some embodiments, the kit may also include multiple doses and may be packaged in amounts sufficient for storage and use by a pharmacy (e.g., a hospital pharmacy and a compounding pharmacy). In certain embodiments, a kit may comprise an administered amount of at least one composition as disclosed herein.
VIII method of treatment
Also provided herein are methods for treating a disease or disorder in a subject, the methods comprising administering to a subject in need thereof an effective amount of a SIRP-a antibody or conjugate described herein. Also provided herein is the use of a SIRP-a antibody or conjugate described herein in the manufacture of a medicament for treating a patient in need of treatment with an oligonucleotide in the conjugate. Also provided are SIRP-a antibodies or conjugates as described herein for use in treating a disease or disorder in a subject in need of treatment with an oligonucleotide in the SIRP-a antibody or conjugate. Also provided are SIRP-a antibodies or conjugates for treating a patient as described herein, comprising administering to the patient an effective amount of a SIRP-a antibody or conjugate. In some embodiments, the subject has or is at risk of having cancer. In some embodiments, the disease or disorder is a viral infection. In some embodiments, the disease or disorder is, for example, immunodeficiency in which immune activation may be beneficial. In some embodiments, the disease or disorder is, for example, an autoimmune and/or inflammatory disease or disorder in which immunosuppression and/or modulation may be beneficial.
In some embodiments of the methods of treating cancer as described herein, the cancer treated with the methods disclosed herein is a solid tumor. In some embodiments, the cancer treated with the methods disclosed herein is a liquid tumor. In some embodiments, the cancer treated with the methods disclosed herein is a solid tumor. In certain embodiments, the cancer treated with the methods disclosed herein is breast cancer, colorectal cancer, lung cancer, head and neck cancer, melanoma, lymphoma, cholangiocarcinoma (cholangiocarcinoma), or leukemia. In some embodiments, the cancer includes, but is not limited to, B cell cancer (e.g., multiple myeloma, waldenstrom's macroglobulinemia), heavy chain diseases (such as alpha chain disease, gamma chain disease, and mu chain disease), benign monoclonal gammaglobulinosis, and immune cell amyloidosis, melanoma, breast cancer, lung cancer, bronchogenic cancer, colorectal cancer, prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, cholangiocarcinoma, small intestine or appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, hematological tissue cancer, and the like. Other non-limiting examples of types of cancers suitable for use in the methods encompassed by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphokaposi's sarcoma, lymphoendotheliosarcoma, synovial carcinoma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma), squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, cystic adenocarcinoma, myelogenous carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, liver carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms's tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, neural tube carcinoma, pharynx, cholangiocarcinoma, glioblastoma, glioma, neuroblastoma, retinoblastoma; leukemias such as acute lymphoblastic leukemia and acute myelogenous leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (hodgkin's and non-hodgkin's), multiple myeloma, waldenstrom's macroglobulinemia and heavy chain diseases. In some embodiments, the cancer is epithelial in nature and includes, but is not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In other embodiments, the epithelial cancer is non-small cell lung cancer, non-papillary renal cell carcinoma, cervical cancer, ovarian cancer (e.g., serous ovarian cancer), or breast cancer. Epithelial cancers may be characterized in a variety of other ways including, but not limited to, serous, endometrium-like, mucinous, clear cells, brenner, or undifferentiated. In certain embodiments, the cancer treated with the methods disclosed herein is selected from the list consisting of: mantle Cell Lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), burkitt's lymphoma (Burkitts lymphoma), multiple Melanoma (MM), chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), small Lymphocytic Lymphoma (SLL), hairy Cell Leukemia (HCL), lymphoplasmacytic lymphoma (LPL), skeletal Myolymphoma (SML), splenic Marginal Zone Lymphoma (SMZL), follicular Central Lymphoma (FCL), colorectal cancer, non-small cell lung cancer (NSCLC), head and neck cancer, breast cancer, pancreatic cancer, glioblastoma (GBM), prostate cancer, esophageal cancer, renal cell carcinoma, liver cancer, bladder cancer, and gastric cancer. In some embodiments, the cancer is lung cancer, cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma), squamous cell carcinoma, brain tumor, glioblastoma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, skin cancer, lung cancer, endometrial cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, urinary tract cancer, urothelial cancer, breast cancer, peritoneal cancer, uterine cancer, salivary gland cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, melanoma, multiple myeloma and B-cell lymphoma, non-hodgkin's lymphoma (NHL), acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), meccell carcinoma, hairy cell leukemia, or Chronic Myelogenous Leukemia (CML), including cancer metastasis thereof. In some embodiments, the cancer is melanoma or renal cancer. In some embodiments, the cancer is a SIRP- α expressing or overexpressing melanoma or renal cancer.
In some embodiments, the cancer or tumor is known or predicted to be unresponsive to a PD-L1 or PD-1 inhibitor (e.g., when administered as monotherapy, or when administered in the absence of an anti-SIRP-a antibody). In some embodiments, an individual to be treated by a method of the disclosure is known or predicted to be unresponsive to PD-L1 or a PD-1 inhibitor (e.g., when administered as monotherapy, or when administered in the absence of an anti-SIRP-a antibody). In some embodiments, the individual does not achieve a significant therapeutic response to PD-L1 or a PD-1 inhibitor (e.g., prior to administration of the conjugate or composition of the invention). In some embodiments, the subject has been treated with PD-L1 or a PD-1 inhibitor prior to administration of the conjugate or composition of the invention. In some embodiments, prior to administration of the conjugates or compositions of the present disclosure, the subject has been treated with PD-L1 or a PD-1 inhibitor and is not responsive to the treatment with PD-L1 or a PD-1 inhibitor (e.g., when administered as monotherapy, or when administered in the absence of an anti-SIRP-a antibody). In some embodiments, the PD-L1 or PD-1 inhibitor is an antibody that binds PD-L1 or PD-1. In some embodiments, the PD-L1 or PD-1 inhibitor is pembrolizumab @Merck), nawuzumab (/ >Bristol Myers Squibb), cimip Li Shan antibody (/ >Regeneron/Sanofi), alemtuzumab (/ >)Genentech) rituximab (/ >GlaxoSmithKline), dewaruzumab (/ >Astrazeneca) or avermectinEMD Serono/Pfizer)。
In some embodiments, the cells of the cancer or tumor express or overexpress human SIRP-a (e.g., on the cell surface thereof), i.e., a SIRP-a positive cancer or tumor. In some embodiments, the cells of the cancer or tumor do not express or overexpress human SIRP- α (e.g., on their cell surfaces), i.e., SIRP- α negative cancer or tumor. In some embodiments, the cells of the cancer or tumor express or overexpress human CD47 (e.g., on the cell surface thereof).
In some embodiments, the methods for treating cancer disclosed herein further comprise administering an additional therapeutic agent, e.g., in combination with a conjugate of the invention. In some embodiments, the additional therapeutic agent includes an immunotherapy (including but not limited to an immune checkpoint inhibitor, such as an anti-PD 1, anti-PD-L1, or anti-CTLA 4 antibody), chemotherapy, radiation therapy, cell-based therapy, an anti-cancer vaccine, or an anti-cancer agent (including but not limited to a therapeutic antibody or other biological agent, or a small molecule inhibitor).
In some embodiments, the methods disclosed herein for treating cancer further comprise administering PD-L1 or a PD-1 inhibitor, e.g., in combination with a conjugate of the present disclosure. In some embodiments, the PD-L1 or PD-1 inhibitor is an antibody, e.g., an anti-PD-L1 antibody or an anti-PD-1 antibody. In some embodiments, the PD-L1 or PD-1 inhibitor is a peptide or small molecule inhibitor. Suitable examples of PD-L1 inhibitors are known in the art and include, but are not limited to, alemtuzumab @Genentech), avermectin (/ >EMD Serono), devaluzumab @AstraZeneca), KN035, CK-301, AUNP, CA-170 and BMS-986189. Suitable examples of PD-1 inhibitors are known in the art and include, but are not limited to, pembrolizumab (/ >)Merck), nawuzumab (/ >Bristol Myers Squibb), cimip Li Shan antibody (/ >Regeneron/Sanofi), rituximab (/ >)GlaxoSmithKline), JTX-4014, swamp mab (spartalizumab) (PDR 001), carlizumab (camrelizumab) (SHR 1210), midodv Li Shan (sintilimab) (IBI 308), tirelimumab (tislelizumab) (BGB-a 317), terlipressin Li Shan (toripalimab) (JS 001), INCMGA00012, AMP-224 and AMP-514. /(I)
In some embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an immunoconjugate described herein, wherein the immunoconjugate binds to SIRP-a, such as a CpG oligonucleotide-antibody immunoconjugate comprising a SIRP-a antibody or an antigen-binding fragment thereof, and wherein the disease or disorder treated is a cancer characterized by SIRP-a overexpression. In some embodiments, such cancers include breast cancer, ovarian cancer, lung cancer, pancreatic adenocarcinoma, colon cancer, hepatocellular carcinoma, bladder cancer, and gallbladder cancer. In some embodiments, the immunoconjugate comprises an oligonucleotide listed in one of table 2 and tables 14 to 17.
In some embodiments, the method of treatment comprises administering a CpG-Ab immunoconjugate that binds to SIRP-a present on bone marrow cells and/or monocytes, and treating to kill or damage tumor cells such that the volume, size, and/or growth of the tumor is reduced or inhibited. In some embodiments, the method of treatment comprises administering a CpG-Ab immunoconjugate that binds SIRP-a on a tumor cell and treats killing or damaging the tumor cell such that the volume, size, and/or growth of the tumor is reduced or inhibited. In some embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a CpG-Ab immunoconjugate described herein, wherein the CpG-Ab immunoconjugate binds to SIRP-a, such as a CpG-Ab immunoconjugate comprising a SIRP-a antibody or antigen-binding fragment thereof, and wherein the disease or disorder treated is a cancer characterized by a tumor cell that expresses SIRP-a. In some embodiments, the disease or disorder treated is a cancer in which tumor cells express SIRP- α. In some embodiments, provided herein are methods for treating a disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of a CpG-Ab immunoconjugate described herein, wherein the CpG-Ab immunoconjugate binds to SIRP-a, such as a CpG-Ab immunoconjugate comprising a SIRP-a antibody or antigen-binding fragment thereof, and wherein the disease or disorder treated is a cancer characterized by a tumor cell that does not express SIRP-a.
In some embodiments, the cancer treated with the methods disclosed herein is resistant to at least one immunotherapy. In some embodiments, the cancer treated with the methods disclosed herein is resistant to at least one cancer therapy selected from the group consisting of chemotherapy, radiation, targeted therapy, vaccine therapy, and CAR-T therapy. In some embodiments, the method of treating cancer comprises co-administering to a subject having cancer (i) a therapeutically effective amount of a CpG-containing immunostimulatory oligonucleotide or a CpG antibody immunoconjugate; and (ii) when the cancer is treated with an immunotherapeutic agent alone, the treated cancer has shown resistance or unresponsiveness to the immunotherapeutic agent. In some embodiments, the treatment comprises a combination treatment with an immunoconjugate of the invention and a therapeutic antibody, a small molecule cancer treatment, a cell therapy, CAR-T, chemotherapy, radiation therapy, or the like.
In certain embodiments, cancers treated with the methods provided herein have been shown to not respond to immune checkpoint modulator treatment. In certain embodiments, the immune checkpoint modulator is a PD-1 inhibitor. In certain embodiments, the immune checkpoint modulator is a PD-L1 inhibitor. In some embodiments, the method of treating cancer comprises co-administering to a subject having cancer (i) a therapeutically effective amount of a CpG-containing immunostimulatory oligonucleotide or a CpG-Ab immunoconjugate; and (ii) a therapeutically effective amount of a PD-1 inhibitor. In some embodiments, the method of treating cancer comprises co-administering to a subject having cancer (i) a therapeutically effective amount of a CpG-containing immunostimulatory oligonucleotide or a CpG-Ab immunoconjugate; and (ii) a therapeutically effective amount of a PD-L1 inhibitor. In particular, in some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the treatment is directed to a subject that is non-responsive or resistant to a PD-1 or PD-L1 inhibitor, and such subject is treated with a CpG-Ab immunoconjugate that binds SIRP-a, such as a CpG-Ab immunoconjugate comprising an anti-SIRP-a antibody or antigen-binding fragment thereof.
In some embodiments, the cancer prevented or treated using the methods provided herein is a recurrent episode of cancer in a subject with partial or complete remission of the previous cancer. In certain embodiments, the prior cancer is a liquid cancer and the recurrent cancer prevented or treated is a liquid cancer. In certain embodiments, the prior cancer is a solid cancer and the recurrent cancer prevented or treated is a solid cancer. In certain embodiments, the prior cancer is a liquid cancer and the recurrent cancer prevented or treated is a solid cancer. In certain embodiments, the prior cancer is a solid cancer and the recurrent cancer prevented or treated is a liquid cancer.
In some embodiments, the cancer prevented or treated using the methods provided herein is a primary cancer recurrence episode in a subject after the subject exhibits partial or complete remission. In some embodiments, the cancer prevented or treated using the methods provided herein is a second cancer recurrence episode in the subject after the subject exhibits partial or complete remission. In some embodiments, the cancer prevented or treated using the methods provided herein is a third cancer recurrence episode in the subject after the subject exhibits partial or complete remission. In some embodiments, the cancer prevented or treated using the methods provided herein is a cancer recurrence episode following a third cancer recurrence episode in the subject after the subject exhibits partial or complete remission.
In some embodiments of the methods and uses described herein, wherein the CpG-containing immunostimulatory oligonucleotide specifically binds to the TLR9 receptor of the target cell after administration of the CpG-SIRP-a-Ab immunoconjugate.
Examples
The subject matter of the present disclosure will be better understood with reference to the following examples, which are provided as exemplary embodiments of the invention and not as limiting examples.
Material
Prototype peptides were prepared internally, but were purchased at conventional peptide suppliers (e.g., CPC SCIENTIFIC). The oligonucleotides are internal or prepared by LGC. The transglutaminase used in these examples was isolated from various bacterial strains of Streptomyces verticillium (e.g., ajinomoto). Q-tag mAbs were generated at Sino Biologicals or internally.
Oligonucleotide production
Oligonucleotides are generally prepared according to the solid phase synthesis procedure shown below, starting with an initial deprotection of the solid support for oligonucleotide synthesis, followed by coupling of the solid support with the first nucleotide, thiolation to give phosphorothioate, and repeated deprotection and coupling to give the entire oligonucleotide sequence.
The general synthesis of oligonucleotides as provided herein is described below.
Deprotection: a controlled pore glass solid support protected with dimethoxytrityl-1, 3-propanediol glycolate (DMTO-C3-CPG,The volume density is 0.26-0.36g/cc, the load is 30-40 mu mol/g, and toluene (v/v) containing 3% dichloroacetic acid is reacted at 25 ℃ to obtain the deprotected CPG carrier. UV absorbance of an aliquot of the reaction mixture was measured to identify the end point of the reaction (using the fixed table command set, wavelength 350nm, target minimum absorbance 0.25 OD) and confirm removal of the dimethoxytrityl protecting group.
Activation/coupling: for the 3 'end, the deprotected CPG vector was coupled to the first nucleotide phosphoramidite precursor by adding the desired 3' nucleotide (3 eq.) to the reactor containing the deprotected CPG vector at 25℃in the presence of the activator 5-ethylsulfanyl-1H-tetrazole (0.5M in ACN) at 60% nucleotide concentration and mixing for 5 minutes.
Thiolation/vulcanization: after the coupling step, the phosphorothioate triester linkage of the added nucleotide precursor is thiolated (or sulfided) by addition of Polyorg Sulfa (3-phenyl-1, 2, 4-dithiazolin-5-one) in anhydrous ACN at 0.15M to give the phosphorothioate.
And (3) end capping: after sulfidation, CPG supports and bound nucleotides were treated with two capping compositions (capping composition A: ACN with 20% N-methylimidazole; capping composition B:20% acetic anhydride, 30% pyridine, 50% ACN) to block unreacted nucleotide reactants.
Repeating synthesis: the remaining nucleotides are added sequentially from the 3 'end to the 5' end by repeating the steps of deprotection, activation/coupling, thiolation/sulphurisation and capping as described above, using phosphoramidite precursors in an appropriate solution, to obtain the desired oligonucleotide sequence in protected form. All phosphoramidite precursors were mixed with CPG support for 5min during the coupling step, except for 15min of dT-thiophosphorous amide.
Selected phosphoramidite precursors used in the synthesis are shown below. The phosphoramidite precursors are prepared in solution with a solvent and at concentrations that respectively show the use to be used in the coupling step.
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Exemplary Fmoc protected oligonucleotide compounds 6.1a, 6.2a and 6.3a obtained from the synthetic steps described above are shown below. Deprotection, purification, and coupling of compound 6.1a to prepare compound 6.1b are described further below.
Fmoc protected CPG Supported Compound 6.1a
Fmoc protected CPG Supported Compound 6.2a
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Fmoc protected CPG Supported Compound 6.3a
CPG supported oligonucleotide compound 6.1a obtained from Fmoc protection synthesized above was cleaved simultaneously from the carrier and deprotected by reacting CPG carrier with ammonium hydroxide containing 20mM dithiothreitol, methylamine 1:1 (v/v) at room temperature for 2 hours to give crude compound 6.1a. The crude product was purified by ion-pair reverse phase HPLC (IP-RP-HPLC) and its identity (identity) was confirmed by ESI-MS. The crude compound 6.1a was purified by HPLC and desalted.
The compound 6.1a was then reacted with O- [2- (Fmoc-amino) -ethyl ] -O' - [3- (N-succinimidyloxy) -3-oxopropyl ] polyethylene glycol (Fmoc-N-amido-dPEG 24 -NHS ester) in sodium bicarbonate buffer to give Fmoc protected compound 6.1b. Fmoc-protected compound 6.1b was reacted with ammonium hydroxide containing 20mM dithiothreitol in methylamine 1:1 (v/v) at room temperature for 2 hours to give crude compound 6.1b. The crude product was purified by ion-pair reverse phase HPLC (IP-RP-HPLC) and its identity was confirmed by ESI-MS. The crude compound 6.1b was purified by HPLC, desalted and lyophilized to give purified oligonucleotide 6.1b.
Antibody production
According to the manufacturer's manual, internally generated antibodies are typically expressed in suspension cultures of the Expi293 system (thermo fisher). The expressed antibodies were purified via protein a capture using MabSelectLX chromatography (GE) eluting with 0.1M citrate (pH 3.3) and dialyzed in a final buffer composition of 1X PBS (phosphate buffered saline, pH 7.4).
Single step conjugation method via mTG (microbial transglutaminase)
A Q-tag having sequence RPQGFGPP (SEQ ID NO: 49) is genetically linked to the C-terminus of the heavy chain of the antibody. For conjugation, the purified antibody (containing an engineered Q tag at the C-terminus of the heavy chain) was first buffer exchanged into 25mM Tris, 150mM NaCl pH 8. The Ab-Q tag containing moiety and CpG were added in a 1:1.3 molar ratio and incubated overnight at room temperature with a final concentration of 1% mTG (w/v) (Ajinomoto). The final concentration of antibody for conjugation is typically about 20-25 μm. The mixture was loaded into Q sepharose HP (GE) equilibrated in 20% buffer B (40 mM Tris, 2M NaCl pH 8) and 80% buffer A (40 mM Tris, pH 8). The column was washed with 5 column volumes of 20% buffer B. The separation was achieved by using a linear gradient of 20% B to 60% B at 30 column volumes. The DAR1 peak fractions (Q tag conjugated to one CpG moiety) were pooled and concentrated followed by a gel filtration step using S200 (GE). The monomeric peak fractions were combined and concentrated.
Biological evaluation of CpG-nucleotides and antibody-CpG nucleotide conjugates
Trima residue was received from Vitalant and diluted 1:4 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare) (underplayed). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface and resuspended in FACS buffer (PBS with 0.5% bovine serum albumin (Gibco)). B cells were purified by negative selection using B cell isolation kit II, human (Miltenyi Biotec) and LS column (Miltenyi Biotec) according to the manufacturer's protocol.
PBMCs were then plated in complete RPMI (rpmi+10% FBS) on a 96-well format (500K/well). Five-fold serial dilutions of 100nM to 6.4pM antibodies and conjugated antibodies and 1uM to 64pM CpG oligonucleotides were added to cells at 37 ℃ at 5% CO 2 for 48 to 96 hours. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:4000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 19, anti-CD 20, anti-CD 40, anti-HLADR and anti-CD 80 (for B cell assays), and anti-CD 14, anti-CD 3, anti-CD 19, anti-CD 14, anti-CD 123, anti-CD 11c and anti-CD 86 (for pDC assays) for 30 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. CountBIght TM absolute count beads (Thermo Fisher) were added to each well to count the number of cells. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Lineage specific cells (CD 19, CD3, CD 14) were first excluded before CD123 +CD11c- cells were gated to identify pDC and CD19 +、CD20+ or CD19 +CD20+ cells were gated to identify B cells.
Example 1: activity of free immunomodulatory oligonucleotides (CpG) in human PBMC
Human PBMC were treated with free CpG (SEQ ID NOs: 3 and 26-28) to evaluate their respective activities as observed by HLADR and CD40 expression on CD19 positive B cells (as shown in FIGS. 1A-1B). CpG (SEQ ID NOS: 26-28) showed all enhanced activity compared to CpG (SEQ ID NO: 3).
Example 2: immunomodulatory oligonucleotides and the activity of their respective antibody conjugates
Various CpG oligonucleotides (SEQ ID NOS: 3-25) were tested for their effect on proliferation and/or activation of B cells. FIGS. 2A-2C show the individual activities of individual selected CpG's. All CpG oligonucleotides tested enhanced B cell activation after 48 hours of incubation. All cpgs increased B cell number and CD40 expression as determined by counting beads to calculate absolute B cell number and CD40 expression. The selected number of CpG oligonucleotides tested showed an enhanced effect on B cell proliferation and activation compared to CpG (SEQ ID NO: 3).
Example 3: transglutaminase mediated conjugation
Transglutaminase mediated conjugation was tested using oligonucleotide A (having the sequence: tucgtcgtgacgtt, SEQ ID NO: 1) coordinated to a pegylated linker (-NH-C (=O) -PEG 23-NH2, structure shown below) and Q tag peptide sequences SEQ ID NO:39-47 and 50-52.
2Nmol of Q tag was added to 1nmol of linker in PBS in the presence of 0.04nmol of transglutaminase. The final concentration of the linker was 50. Mu.M. The reaction was kept at room temperature and quenched with 8M formamide at 1 hour. The reaction solution was analyzed at 60 ℃ using reverse phase HPLC with Xbridge C18 column (4.6×150 mM) using solvent a (50 mM TEAA in water) and solvent B (acetonitrile), with a gradient of 20% to 60% solvent B for 10 minutes. Alternatively, the reaction solution was analyzed at 50 ℃ using reverse phase HPLC with a Luna 3 μc18 column (4.6x50 mm) using solvent a (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile), with a gradient of 10% to 70% solvent B for 10 minutes.
Figure 3 shows the yields of transglutaminase mediated conjugation and peptide deamidation with various Q tags. RPQGF (SEQ ID NO: 47), RPQQF (SEQ ID NO: 46), RPRPQQF (SEQ ID NO: 50) show a high percentage of conjugates and suitably low deamidation.
FIGS. 4A-4B show conjugation and de-conjugation over time of two conjugates prepared from Q-tags with SEQ ID NOS: 39 and 47. At all Q-tag to linker+CpG ratios tested, RPQGF (SEQ ID NO: 47) had a higher percentage of conjugation over a 16 hour duration. In addition, the rate of decoking was also slower for RPQGF (SEQ ID NO: 47) compared to LSLSPGLLQGG (SEQ ID NO: 39).
Example 4: assessment of Activity of free CpG on CD40 expression by CD19+ B cells
Materials and methods
Trima residue was received from Vitalant and diluted 1:2 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (rpmi+10% FBS).
PBMCs were then plated in full RPMI on a 96-well format (500K/well). Five-fold serial dilutions of 1uM to 64pM CpG oligonucleotides were added to cells at 37 ℃ at 5% CO 2 for 48 hours. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:4000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 19, anti-CD 20, anti-CD 40, anti-HLADR and anti-CD 80 for 30 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. B cells were identified as cd19+cd20+ cells and the level of activation markers was assessed by median fluorescence intensity.
For Ramos NFkb reporter assays, ramos-Blue cells NF-kB/AP-1 reporter B lymphocytes were purchased from Invivogen. Cells were grown and maintained in complete DMEM supplemented with 2mM L-glutamine, 10% FBS, 100ug/mL Normacin, penicillin-streptomycin (Pen-Strep), 100ug/mL gecomycin (Zeocin). Ramos-Blue cells were stimulated. Briefly, cells were washed in growth medium without antibiotics. Cells were counted and resuspended at a density of 2×10 6 cells/ml in fresh complete DMEM without selection antibiotic. 20uL of 10uM CpG7-7, cpG 12070 and ODN2006 were added 1:5 titrated into flat bottom 96 well plates, adding 180uL of cell suspension to a final concentration of 1uM to 64pM CpG. Plates were incubated in a 5% CO 2 incubator at 37℃for 24 hours. On the day of assay, QB reagent and QB buffer were thawed prior to use. Quanti-Blue solution was prepared by adding 1mL of QB reagent and 1mL of QB buffer to 98mL of sterile water in a sterile glass bottle. 180uL Quanti-Blue solution was dispensed per well into a new flat bottom 96-well plate. 20uL of supernatant from treated Ramos-Blue cells was then added to a 96-well plate. Plates were then incubated for 6 hours. Optical density was measured at OD655 using a plate reader (Molecular Devices) and the data tabulated in GRAPHPAD PRISM 9.0.
Results
Human PBMCs were treated with free CpG to assess their individual activity as observed by CD40 expression on CD19 positive B cells. As shown in FIG. 5, the series 7CpG (SEQ ID NOS: 29, 30 and 32-36) all showed enhanced activity compared to CpG 12070 (SEQ ID NO: 3).
In NFkb reporter assays, cpG oligomers 7-7, 12070 were compared to ODN2006 (5'-tcgtcgttttgtcgttttgtcgtt-3'; SEQ ID NO: 167). As shown in fig. 6, cpG 7-7 showed significantly higher activity compared to 12070 and ODN 2006.
Example 5: evaluation of CpG Activity on PBMC from different donors
The activities of CpG oligomers 7-6, 7-7 and 12070 in PBMC cells from three different donor systems (D559, D804 and D643) were compared as observed by CD40 expression. The activity evaluation of the CpG oligomer was performed using the same method as in example 16 above.
The results showed that the higher activity of 7-6 and 7-7 compared to 12070 was not dependent on the donor (FIGS. 7A-7C).
Example 6: contribution of 5 'bromo 2' deoxyuridine and PEG linkage to CpG Activity
For evaluation of CpG oligonucleotides in human PBMC, trima residues were received from Vitalant and diluted 1:4 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare) (underplayed). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface and resuspended in FACS buffer (PBS with 0.5% bovine serum albumin (Gibco)). PBMCs were then plated in complete RPMI (rpmi+10% FBS) on a 96-well format (500K/well). Five-fold serial dilutions of 1uM to 64pM CpG oligonucleotides were added to cells at 37 ℃ at 5% CO 2 for 48 to 96 hours. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:4000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 19, anti-CD 40 and anti-CD 86 for 30 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Cd19+, cd20+ or cd19+cd20+ cells were gated to identify B cells. The data was tabulated using GRAPHPAD PRISM 9.0.0.
As shown in FIG. 8, the non-bromo modified CpG oligonucleotides 9-9 and 9-10 activated CD86 expression at the 5' uridine. This suggests that bromine modification is not an essential component of the respective oligonucleotides.
Example 7: activation of human monocytes and dendritic cells with anti-SIRP-alpha antibody-CpG oligonucleotide conjugates
Activation of monocytes and dendritic cells by anti-SIRP-alpha antibody-CpG oligonucleotide conjugates was assessed. Human Peripheral Blood Mononuclear Cells (PBMCs) were treated with the conjugate and compared to unconjugated sirpa antibodies.
Experiments using anti-SIRP-alpha antibody-CpG oligonucleotide conjugates utilized 7-7CpG oligonucleotides (SEQ ID NO: 35) conjugated to anti-SIRP-alpha antibodies comprising: (a) the heavy chain sequence of SEQ ID NO. 68 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 1), (b) the heavy chain sequence of SEQ ID NO. 66 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 4), or (c) the heavy chain sequence of SEQ ID NO. 67 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 1 AAA). Trima residue was received from Vitalant and diluted 1:3 with phosphate buffered saline (PBS, gibco). Diluted blood was desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (rpmi+10% FBS). PBMCs were then plated onto 96-well format (1 e 6/well) in complete RPMI. Triple serial dilutions of anti-SIRP-alpha antibody-CpG oligonucleotide conjugate and unconjugated anti-SIRP-alpha antibody were added to cells at 37℃from 300nM to 0.41nM for 24 hours at 5% CO 2. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:5000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagents (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 3, anti-CD 19, anti-CD 56, anti-CD 16, anti-HLADR, anti-CD 40, anti-CD 86, anti-CD 304, anti-CD 1c and anti-CD 141 (Thermo Fisher, biolegend) for 60 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Monocytes were identified as CD3 -CD19-CD14+ cells. CD11c high dendritic cells were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+. Conventional DCs were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+CD1c+ and plasmacytoid DCs were identified as CD3 -CD19-CD56-CD14-CD11c-HLADR+CD304+. The level of activation markers was assessed by median fluorescence intensity.
As shown in fig. 10A-10D, the anti-SIRP-a antibody-CpG oligonucleotide conjugates exhibited robust activation of CD86 on human monocytes, conventional dendritic cells (cDC 1), CD11c high dendritic cells, and plasmacytoid DCs (pDC), respectively. The activity of the conjugate was significantly higher than the unconjugated anti-SIRP-a antibody.
Example 8: anti-SIRP-alpha antibody-CpG oligonucleotide conjugate stimulated IRF7 and IL-6 induction
Stimulation of interferon regulatory factor 7 (IRF 7) and interleukin-6 (IL-6) in bone marrow cells by anti-SIRP-alpha antibody-CpG oligonucleotide conjugates was evaluated. PBMCs were treated with conjugate and compared to unconjugated anti-SIRP-a antibodies.
Experiments using anti-SIRP-alpha antibody-CpG oligonucleotide conjugates utilized 7-7CpG oligonucleotides (SEQ ID NO: 35) that bind to anti-SIRP-alpha antibodies comprising: (a) The heavy chain sequence of SEQ ID NO. 68 and the light chain sequence of SEQ ID NO. 73 (for anti-hIgG 1), (b) the heavy chain sequence of SEQ ID NO. 66 and the light chain sequence of SEQ ID NO. 73 (for anti-hIgG 4); and 12070 (SEQ ID NO: 3) conjugated to an anti-SIRP-alpha antibody comprising the heavy chain sequence of SEQ ID NO:67 and the light chain sequence of SEQ ID NO:73 (for anti-hIgG 1 AAA). Trima residue was received from Vitalant and diluted 1:2 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (rpmi+10% FBS). PBMCs were then plated onto 96-well format (1 e 6/well) in complete RPMI. Five-fold serial dilutions of anti-SIRP-a antibody-CpG oligonucleotide conjugate and unconjugated anti-SIRP-a antibody were added to cells at 37 ℃ at 5% CO 2, from 100nM to 6.4pM for 48 hours. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:4000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 3, anti-CD 19, anti-CD 56, anti-CD 16, anti-HLADR, anti-CD 40, anti-CD 86 (Thermo Fisher, biolegend) for 30 min at 4 ℃. The cells were centrifuged and washed twice in FACS buffer. The cells were then treated with transcription factor fixation/permeation concentrate and diluent (eBioscience) for intracellular staining. Briefly, cells were incubated in fresh fixation buffer by mixing 1 part fixation/permeation concentrate with 3 parts fixation permeation diluent. The samples were incubated at 4℃for 30-60 minutes in the absence of light. The sample was then centrifuged at 600g for 5 minutes at room temperature. The pellet was resuspended with 1 Xpermeation buffer, followed by two rounds of washing and centrifugation at 600g for 5 minutes at room temperature. The pellet was resuspended in 100uL of permeation buffer and stained with anti-IL 6, anti-IRF 7 for 60 min at room temperature. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Monocytes were identified as CD3 -CD19-CD14+ cells and dendritic cells were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+. The level of activation markers was assessed by median fluorescence intensity, and cytokine/chemokine expression was assessed as% of CD14 + or CD11c + cells.
As shown in fig. 11A-11D, anti-SIRP-a antibody-CpG oligonucleotide conjugates exhibited robust induction of bone marrow-derived IRF7 and IL6 in human monocytes and dendritic cells. The activity of the conjugate was significantly higher than the unconjugated anti-SIRP-a antibody.
Example 9: anti-SIRP-alpha antibody-CpG oligonucleotide conjugate for enhancing cytokine secretion
The effect of anti-SIRP-a antibody-CpG oligonucleotide conjugates on cytokine secretion in PBMCs was assessed.
Experiments using anti-SIRP-alpha antibody-CpG oligonucleotide conjugates utilized 7-7CpG oligonucleotides (SEQ ID NO: 35) conjugated to anti-SIRP-alpha antibodies comprising: (a) the heavy chain sequence of SEQ ID NO. 68 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 1), (b) the heavy chain sequence of SEQ ID NO. 66 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 4), or (c) the heavy chain sequence of SEQ ID NO. 67 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 1 AAA). Trima residue was received from Vitalant and diluted 1:2 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (rpmi+10% FBS). PBMCs were then plated onto 96-well format (1 e 6/well) in complete RPMI. Five-fold serial dilutions of anti-SIRP-a antibody-CpG oligonucleotide conjugate and unconjugated anti-SIRP-a antibody were added to cells at 37 ℃ at 5% CO 2, from 100nM to 6.4pM for 48 hours. Cells were pelleted by centrifugation at 400×g for five minutes and cytokines in the supernatant were collected and quantified using a LEGENDplex TM Human Inflammation Panel I bead-based immunoassay (catalog No. 740809) of BioLegend according to manufacturer's recommendations. Briefly, the supernatant was plated in assay buffer prior to the addition of cytokine-specific capture beads. Plates were incubated at room temperature for 2 hours, followed by two washes, after which biotinylated detection antibody was added for 1 hour. PE-bound streptavidin was added to the plate without washing and incubated for 30 min at room temperature, followed by two washes, before buffer addition. The assay plates were then processed by flow cytometry using a Attune NxT cell counter (Thermofisher). The data was analyzed with FlowJo 10.6 software (BD) and tabulated using GRAPHPAD PRISM.
As shown in fig. 12A-12D, the level of cytokine secretion in anti-SIRP-a antibody-CpG oligonucleotide conjugate stimulated human PBMC was higher than that of unconjugated anti-SIRP-a antibody. These cytokines include IFN- α2, IFN- γ, IL-6 and IL-10.
Example 10: unconjugated CpG oligonucleotides activate bone marrow cells
The ability of CpG oligonucleotides to activate CD40 in bone marrow cells was compared. Two unconjugated oligonucleotides CpG 7-7 (SEQ ID NO: 35) and CpG-12070 (SEQ ID NO: 3) were tested.
Trima residue was received from Vitalant and diluted 1:3 with phosphate buffered saline (PBS, gibco). Diluted blood was desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (rpmi+10% FBS). PBMCs were then plated onto 96-well format (1 e 6/well) in complete RPMI. The CpG oligonucleotides were incubated with 40nM concentration of cells at 37℃for 48 hours at 5% CO 2. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:5000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 3, anti-CD 19, anti-CD 56, anti-CD 16, anti-HLADR, anti-CD 40 and anti-CD 86 (Thermo Fisher, biolegend) for 60 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Monocytes were identified as CD3 -CD19-CD14+ cells. Dendritic cells were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+. The level of activation markers was assessed by median fluorescence intensity.
As shown in FIG. 13, the 7-7CpG oligonucleotides showed higher CD40 activation in human bone marrow cells compared to the 12070CpG oligonucleotides.
Example 11: anti-SIRP-alpha antibody-CpG oligonucleotide conjugates exhibiting tumor phagocytosis of monocyte-derived M2 macrophages via CD 47-SIRP-alpha blockade
The ability of the anti-SIRP-a antibody-CpG oligonucleotide conjugate to stimulate tumor phagocytosis of monocyte-derived M2 macrophages compared to unconjugated anti-SIRP-a antibody or medium control was assessed.
Experiments using anti-SIRP-alpha antibody-CpG oligonucleotide conjugates utilized 7-7CpG oligonucleotides (SEQ ID NO: 35) conjugated to anti-SIRP-alpha antibodies comprising: the heavy chain sequence of SEQ ID NO. 66 and the light chain sequence of SEQ ID NO. 73 (for anti-hIgG 4). Human CD14 + cells were purified from Trima residue (Vitalant) using Ficoll-Paque Plus and negative selection (monocyte isolation kit II (Monocyte Isolation Kit II), miltenyi Biotec) according to the manufacturer's protocol. Monocyte-derived macrophages (MDM) were prepared by seeding 1 million CD14 + cells into a growth medium supplemented with 10% FBS and 50ng/mL MCSF in a 150mm tissue culture dish (Corning). The cells were cultured for 7-11 days. Adherent cells were detached from the plates using TRYPLE SELECT (Thermo FISHER SCIENTIFIC). Target cells (DLD-1) were labeled with CELLTRACE CFSE cell proliferation kit (Thermo FISHER SCIENTIFIC) according to the manufacturer's instructions. 100,000 target cells and 50,000 MDMs were incubated with 150nM unconjugated anti-sirpa h igg4 and corresponding CpG conjugates in ultra-low attachment U-bottom 96-well plates (Corning) for 2 hours at 37 ℃. For flow cytometry, cells were incubated in human FcR blocking reagent (Miltenyi Biotec) and stained with fluorochrome-labeled anti-CD 33 antibodies. To eliminate macrophage/target cell adhesion from the assay, anti-CD 326 antibodies were included. In addition, a pulse geometry gate of forward scatter signal area and height is used to select single cells. Fixable viability dye (Thermo FISHER SCIENTIFIC) was used to identify living cells. Cells were obtained on FACS Canto II flow cytometer (BD Biosciences) and subsequently analyzed using FlowJo software. The percentage phagocytosis indicates the percentage of viable CD33 + macrophages that were negative for CD326 staining and positive for CFSE staining.
As shown in fig. 14, the anti-SIRP-a antibody-CpG oligonucleotide conjugate showed a robust stimulation of DLD-1 tumor cell phagocytosis by M2 macrophages equivalent to unconjugated antibody, confirming the ability of the conjugate to maintain complete CD 47-SIRP-a blockade.
Example 12: anti-tumor Activity of anti-SIRP-alpha antibody-CpG oligonucleotide conjugates
Anti-SIRP-a antibody-CpG oligonucleotide conjugates were constructed with either mouse IgG2a or mouse IgG1 Fc domains and compared for anti-tumor activity in SIRP-a positive syngeneic tumor model RENCA.
Experiments with anti-SIRP-alpha antibody-CpG oligonucleotide conjugates utilized a mouse CpG oligonucleotide 4523 (SEQ ID NO: 121) conjugated to an anti-SIRP-alpha antibody comprising a heavy chain comprising a sequence of DVQLVESGGGVVRPGESLRLSCAASGFTFSSNAMSWVRQAPGKGLEWLAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNTLTAEDTAVYYCARETWNHLFDYWGLGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGRPQGFGPP(SEQ ID NO:90)( for mIgG2 a) or DVQLVESGGGVVRPGESLRLSCAASGFTFSSNAMSWVRQAPGKGLEWLAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNTLTAEDTAVYYCARETWNHLFDYWGLGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFIYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGRPQGFGPP(SEQ ID NO:91)( for mIgG 1) and a light chain comprising a sequence of ALTQPASVSANPGETVKIACSGGDYYSYYYGWYQQKAPGSALVTVIYSDDKRPSDIPSRFSGSASGSTATLTITGVRAEDEAVYYCGGYDYSTYANAFGAGTTLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS(SEQ ID NO:111). 4523 murine CpG oligonucleotide has the sequence tucgtcgtgacgtt-c3, where the lower case letters indicate phosphorothioate linkages, the bold indicates iodo-uridine, and the underlined indicates the phosphotriester linker (SEQ ID NO: 121). RENCA kidney cancer cells (ATCC) were cultured in complete RPMI 1640 (RPMI 1640+10% FBS (Gibco)) at 37℃and 5% CO 2. Cells were detached with trypsin 0.25% (Gibco) and washed twice with RPMI 1640 (Gibco). Cells were resuspended in RPMI 1640 at 20E6/mL and kept on ice until use. 100uL of suspended cells were subcutaneously implanted into the right flank of 6 week old female BALB/c mice (CHARLES RIVER). Tumor size was measured with a caliper one week and recorded twice after approximately 35 days after 9 days of implantation until the duration of the study. Tumor volume was calculated using the following formula: (length x width)/2. Once the tumors reached an average of 75mm 3, approximately 3 days post-implantation, mice were randomly grouped according to tumor size and treatment started. 3 doses of conjugate were administered intraperitoneally every 3 days at 3mg/kg or 10 mg/kg. Mice with tumors exceeding 2,000mm 3 or exhibiting any signs of distress were humanly sacrificed at any time during the study, according to IACUC approved animal protocols.
As shown in fig. 15A and 15B, the anti-SIRP-a antibody-CpG oligonucleotide conjugate showed an equivalent anti-tumor response at a lower dose of 3 mg/kg. However, at higher dose levels of 10mg/kg, the igg2a Fc-containing conjugates showed reduced anti-tumor inhibition compared to the igg1 Fc domain-containing conjugates. The anti-SIRP-a antibody-CpG oligonucleotide conjugate provides robust single agent activity against one poorly immunogenic tumor type RENCA. Without wishing to be bound by theory, it is believed that the differential response found at the higher dose level of mIgG2a may be due to scorpion action (see, e.g., kurlander RJ. (1983) J immunol.131 (1): 140-7 and Voets, e.et al (2019) j.immunotherapeutic cancer 7,340), effector cell type depletion, or a combination of both.
Example 13: monocyte activation of anti-SIRP-alpha antibody-CpG oligonucleotide conjugates in PBMC co-cultured with tumor cell lines
The ability of anti-SIRP-alpha antibody-CpG oligonucleotide conjugates to activate monocytes when co-cultured in the presence of SIRP-alpha positive or SIRP-alpha negative tumor cell lines was assessed.
Experiments using anti-SIRP-alpha antibody-CpG oligonucleotide conjugates utilized 7-7CpG oligonucleotides (SEQ ID NO: 35) conjugated to anti-SIRP-alpha antibodies comprising: (a) the heavy chain sequence of SEQ ID NO. 68 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 1), (b) the heavy chain sequence of SEQ ID NO. 66 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 4), or (c) the heavy chain sequence of SEQ ID NO. 67 and the light chain sequence of SEQ ID NO. 75 (for anti-hIgG 1 AAA). Trima residue was received from Vitalant and diluted 1:2 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (rpmi+10% FBS). PBMCs were then plated onto 96-well format (0.5 e 6/well) in complete RPMI. Tumor cells (0.025E6/well) from either the labeled DLD-1 parent CFSE or DLD-1 transduced to overexpress SIRP-alpha+ and GFP were added to PBMC at 20:1 (effector: target). anti-SIRP-alpha antibody-CpG oligonucleotide conjugates and unconjugated anti-SIRP-alpha antibodies were added to cells at 37℃from 300nM to 0.41nM for 48 hours at 5% CO 2. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:4000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 3, anti-CD 19, anti-CD 56, anti-CD 16, anti-HLADR, anti-CD 40 and anti-CD 86 for 30 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Monocytes were identified as CD3 -CD19-CD14+ cells and dendritic cells were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+. The level of activation of CD14 + monocytes by the activation markers was assessed by median fluorescence intensity.
Figures 16A and 16B show data for three SIRP-a-CpG oligonucleotide conjugates each conjugated to a different Fc domain (human IgG4, human IgG1 and human IgG 1-AAA). The hIgG4 conjugate showed the strongest CD40 induction in monocytes when co-cultured with DLD-1 transduced to overexpress SIRP-alpha and the parent DLD-1 not expressing SIRP-alpha compared to the other conjugates and unconjugated antibodies. CD40 induction in monocytes in the presence of DLD-1 independent of SIRP-alpha expression suggests that bone marrow activation may be achieved via direct engagement and/or other regulatory mechanisms of SIRP-alpha expressed on the CD14 + monocyte population.
Example 14: anti-SIRP-alpha antibody-CpG oligonucleotide conjugate as TLR9 agonist for activating bone marrow cell and promoting anti-tumor immunity
Novel therapies that combine both innate and adaptive immune responses can result in more robust and durable anti-cancer immunity (Kobold et al (2019) Proc.Natl. Acad. Sci.116:1087-1088). Activation of toll-like receptor 9 (TLR 9) by unmethylated CpG Oligonucleotides (ODN) promotes the innate inflammatory response and induction of adaptive immunity (Dowling et al (2016) Clin. Transl. Immunol.5:e 85-10). Several clinical benefits of CpG-ODN have been demonstrated in intratumoral injection melanoma patients (Hamid et al (2019) The Oncol.25:343-359). Signal regulatory protein alpha (SIRPalpha) is a myelosuppressive receptor that inhibits immune activation after binding to its ligand CD47 (Kuo et al (2020) J.Hematol. Oncol. 13:160). The clinical benefit of blocking the CD47-SIRP alpha bone marrow checkpoint pathway has been demonstrated in solid tumor patients (Lee et al ALX148,a CD47 blocker in combination with standard chemotherapy and antibody regimens in patients with gastric/gastroesophageal junction(GC)cancer and head and neck squamous cell carcinoma(HNSCC);ASPEN-01.SITC 2020).
Examples 14-22 provide preclinical data demonstrating that anti-SIRP-a antibody-CpG oligonucleotide conjugates deliver differentiated TLR-9 agonists (T-CpG) to bone marrow cells via SIRP-a and fcγr engagement, triggering TLR9 signaling, cell activation and immunomodulation, resulting in robust anti-tumor efficacy. The anti-SIRP-a antibody-CpG oligonucleotide conjugates provided significantly higher activity than either of the individual components (i.e., the anti-SIRP-a antibody alone or the CpG oligonucleotide alone) confirming the surprising benefit of combining these selected components.
The anti-SIRP-a antibody-CpG oligonucleotide conjugate contained a differentiated TLR-9 agonist (T-CpG) conjugated to a SIRP-a antibody (fig. 17A). Human SIRP- α is expressed on dendritic cells and bone marrow cells, but not B cells, while TLR9 is expressed on dendritic cells, bone marrow cells and B cells.
Example 15: T-CpG is a potent TLR9 agonist with cross species targeted activation
Materials and methods
Trima residue was received from Vitalant and diluted 1:3 with phosphate buffered saline (PBS, gibco). Diluted blood was desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (RPMI, 10% fbs, 1 xpen/Strep, 1 x Glutamax). PBMCs were then plated onto 96-well format (1 e 6/well) in complete RPMI. Triple serial dilutions of anti-SIRP-alpha antibodies (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO: 75) and anti-SIRP-alpha conjugates (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO:75, conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35)) were added to cells from 300nM to 0.4nM. Free CpG 7-7 (SEQ ID NO: 35) was titrated from 3uM to 4.12 nM. Cells were incubated at 37℃for 24 hours at 5% CO 2. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:5000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagents (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 3, anti-CD 19, anti-CD 56, anti-CD 16, anti-HLADR, anti-CD 40, anti-CD 86, anti-CD 304, anti-CD 1c and anti-CD 141 (Thermo Fisher, biolegend) for 60 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Monocytes were identified as CD3 -CD19-CD14+ cells. Dendritic cells were identified as CD3-CD19-CD56-CD14-HLADR+CD11c+. Conventional DCs were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+CD1c+ and plasmacytoid DCs were identified as CD3 -CD19-CD56-CD14-CD11c-HLADR+CD304+. The level of activation markers was assessed by median fluorescence intensity.
Cynomolgus whole blood was received from BioIVT and diluted 1:3 with phosphate buffered saline (PBS, gibco). Diluted blood was desalted with 15mL of 90% Ficoll-Paque (GE Healthcare) PBS solution. The tube was centrifuged at 400 Xg for 30 minutes. Cells were collected from the interface, resuspended in FACS buffer (PBS (Gibco) containing 0.5% bovine serum albumin) and washed. After one wash, cells were resuspended in ACK lysis buffer (Gibco) for 15 min at room temperature, washed in FAC buffer and centrifuged before repeating. Purified PBMC were resuspended in complete RPMI (RPMI, 10% FBS, 1 XPen/Strep, 1 XPlutamax). PBMCs were then plated onto 96-well format (0.3 e 6/well) in complete RPMI. A triple serial dilution of 1uM to 1.4nM CpG oligonucleotide and 300nM to 0.4nM anti SIRP-alpha antibody (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO: 75) and corresponding antibody CpG conjugate (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO:75, conjugated to CpG oligonucleotide 7-7 (SEQ ID NO: 35)) was added to cells at 37℃for 48 hours. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:5000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 123, anti-CD 3, anti-CD 20, anti-CD 16, anti-CD 8, anti-HLADR, anti-CD 40, anti-CD 69 and anti-CD 86 (thermofisher, biolegend) for 60 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. The level of activation marker identified by dendritic cells as CD3-CD20-CD16-CD14-CD8-CD123-HLADR+CD11c+. was assessed by median fluorescence intensity.
The spleens of mice were treated in ice-cold PBS to single cell suspensions, solubilized with ACK lysis buffer (Gibco), washed twice and resuspended in PBS supplemented with 2% FBS. 1-2 aliquots of 10 6 cells were stimulated in complete RPMI (RPMI+10% FBS,1 XPen/Strep, 1 XPlutamax) for 48 hours. Spleen cells were plated onto 96-well format (1 e 6/well) in the presence of: an anti-SIRP-alpha antibody comprising a heavy chain comprising the sequence of SEQ ID NO. 67 and a light chain comprising the sequence of SEQ ID NO. 73 at 100nM to 6.4 pM; an anti-SIRP-alpha antibody-CpG oligonucleotide conjugate comprising a heavy chain comprising the sequence of SEQ ID NO:67 and a light chain comprising the sequence of SEQ ID NO:73 conjugated to CpG oligonucleotide 4523 (SEQ ID NO: 121); or 1uM to 64pM mT-CpG 4523 titration 1:5. The cells were then incubated at 37℃for 48 hours at 5% CO 2. Cells were pelleted by centrifugation at 400×g for five minutes and stained in Fixable Viability Dye eFluor780 (Thermo Fisher) diluted 1:5000 in PBS at 4 ℃ followed by staining with mouse Fc-blocks (Biolegend) and then with the following antibodies for at least 30 minutes at 4 ℃): anti-SIGLEC H, anti-CCR 7, anti-CD 86, anti-MHCII, anti-GR-1, anti-33D 1 clone 33D1, anti-CD 11b, anti-CD 11c. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). All mobile antibodies were purchased from Biolegend or Thermo Fisher. Dead cells were excluded by gating on eFluor780 negative population. Dendritic cells were identified as CD11c +MHCII+33D1+ or CD11c +MHCII+CD8+. The level of activation markers was assessed by median fluorescence intensity.
Results
Human PBMC (fig. 17B), cynomolgus PBMC (fig. 17C) or mouse splenocytes (fig. 17D) were stimulated with anti-hsrp-a antibodies, anti-hsrp-a antibody-CpG oligonucleotide conjugates, cpG7-7 oligonucleotides or anti-mSIRP-a antibody-CpG oligonucleotide conjugates with murine-reactive mT-CpG for 24 hours or 48 hours and surface marker expression (CD 40) on dendritic cells was determined by flow cytometry. The results demonstrate that CpG7-7 oligonucleotides are potent TLR9 agonists with trans-species targeted dendritic cell activation. In addition, in human dendritic cells, anti-hsrp-a antibody-CpG oligonucleotide conjugates showed significantly increased activation compared to the antibody or oligonucleotide alone.
Example 16: cell type specificity and activation of anti-SIRP-alpha antibody conjugates
The ability of anti-SIRP-a antibody conjugates to specifically activate human target cells expressing both sirpa and TLR9 was tested.
Materials and methods
Trima residue was received from Vitalant and diluted 1:3 with phosphate buffered saline (PBS, gibco). Diluted blood was desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (RPMI, 10% fbs, 1 xpen/Strep, 1x Glutamax). PBMCs were then plated onto 96-well format (1 e 6/well) in complete RPMI. 100nM of an anti-hIgG 4 SIRP-alpha conjugate (an anti-SIRP-alpha antibody-CpG oligonucleotide conjugate having a heavy chain comprising the sequence of SEQ ID NO:66 and a light chain comprising the sequence of SEQ ID NO:73, conjugated to CpG oligonucleotide 7-7 (SEQ ID NO: 35)) or an anti-CD 22 antibody-CpG oligonucleotide conjugate was added to the cells. The anti-CD 22 antibody has a heavy chain comprising a VH domain comprising the sequence QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS(SEQ ID NO:112) with human IgG1 Fc and a VL domain comprising the sequence DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIK(SEQ ID NO:113) and is conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35).
The cells were then incubated at 37℃for 24 hours at 5% CO 2. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:5000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 14, anti-CD 3, anti-CD 19 and anti-CD 86 (Thermo Fisher, bioleged) for 30-60 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor 780 negative population. Monocytes were identified as CD3 -CD19-CD14+ cells. B cells were identified as CD3 -CD19+. The level of activation markers was assessed by median fluorescence intensity.
Results
The antibody conjugates were tested for their ability to specifically activate human target cells. Human PBMCs were stimulated with anti-SIRP-a antibody-CpG oligonucleotide conjugate or anti-CD 22 antibody-CpG oligonucleotide conjugate for 24 hours and CD86 surface marker expression on monocytes (fig. 18A) and B cells (fig. 18B) was determined by flow cytometry. The anti-SIRP-alpha antibody-CpG oligonucleotide conjugate specifically targets and activates SIRP-alpha expressing monocytes, but does not target and activate B cells that do not express SIRP-alpha. In contrast, anti-CD 22 antibody-CpG oligonucleotides targeting B cells exhibited efficient B cell activation.
Example 17: ability of anti-SIRP-alpha antibody conjugates to activate dendritic cells co-cultured with SIRP-alpha positive or SIRP-alpha negative tumor cells
Materials and methods
Trima residue was received from Vitalant and diluted 1:2 with phosphate buffered saline (PBS, gibco). The diluted blood was split into two tubes and desalted with 15mL Ficoll-Paque (GE Healthcare). The tube was centrifuged at 400 Xg for 30 minutes. PBMCs were collected from the interface, resuspended in FACS buffer (PBS containing 0.5% bovine serum albumin (Gibco)) and washed. After one wash, PBMCs were resuspended in complete RPMI (RPMI, 10% FBS, 1 xpen/Strep, 1 x Glutamax). PBMCs were then plated into full RPMI on 96-well format (0.5 e 6/well). Tumor cells (0.025E6/well) from either the labeled DLD-1 parent CFSE or DLD-1 transduced to overexpress SIRP-alpha+ and GFP were added to PBMC at 20:1 (effector: target). The antibodies and corresponding antibody conjugates tested were: an anti-SIRP-alpha hIgG4 antibody-CpG oligonucleotide conjugate (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO:73, conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35)) and unconjugated anti-SIRP-alpha hIgG4 (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO: 73), an anti-SIRP-alpha hIgG1 antibody-CpG oligonucleotide conjugate (heavy chain comprising the sequence of SEQ ID NO:68 and light chain comprising the sequence of SEQ ID NO:75, conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35)) and unconjugated anti-alpha hIgG1 (heavy chain comprising the sequence of SEQ ID NO:68 and light chain comprising the sequence of SEQ ID NO: 75), and an anti-SIRP-alpha hIgG1AAA antibody-CpG oligonucleotide conjugate (heavy chain comprising the sequence of SEQ ID NO:67 and light chain comprising the sequence of SEQ ID NO:75, conjugated to light chain comprising the sequence of SEQ ID NO: 7-alpha hIgG1 (SEQ ID NO: 35)). Antibodies and corresponding antibody-oligonucleotide conjugates were added to cells at 37 ℃ at 5% CO 2, from 300nM to 0.41nM, for 48 hours. Cells were pelleted by centrifugation at 400 Xg for five minutes and stained in Fixable Viability Dye eFluor (Thermo Fisher) diluted 1:4000 in PBS at 4 ℃. Cells were centrifuged and stained in FACS buffer containing FcR blocking reagent (Miltenyi Biotec), anti-CD 14, anti-CD 11c, anti-CD 3, anti-CD 19, anti-CD 56, anti-CD 16, anti-HLADR, anti-CD 40 and anti-CD 86 for 30 min at 4 ℃. Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT flow cytometer (Thermo Fisher) followed by data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on eFluor780 negative population. Dendritic cells were identified as CD3 -CD19-CD56-CD14-HLADR+CD11c+. The level of dendritic cell activation of the activation markers was assessed by median fluorescence intensity.
Results
Human PBMC were co-cultured for 48 hours in the presence of either individual anti-SIRP-alpha antibody-CpG oligonucleotide conjugates or anti-SIRP-alpha antibodies in the presence of DLD-1 cells that overexpressed SIRP-alpha (FIG. 19) or parent DLD-1 cells that did not express SIRP-alpha (FIG. 20). Surface marker (CD 86) expression was determined by flow cytometry. These results demonstrate that sirpa expression on tumor cells significantly enhances activation of bone marrow cells (e.g., dendritic cells) by lower concentrations of anti-SIRP-a antibody-CpG oligonucleotide conjugates in a co-culture assay compared to SIRP-a negative tumor cells. This was observed on all anti-SIRP-a antibody-CpG oligonucleotide conjugates tested and demonstrated that the anti-SIRP-a antibody conjugates can provide lower threshold bone marrow cell activation in the case of SIRP-a positive tumor cells compared to SIRP-a negative tumor cells.
Example 18: ability of anti-SIRP-alpha antibody conjugates to promote phagocytosis of SIRP-alpha positive or SIRP-alpha negative tumor cells
Materials and methods
Human CD14 + cells were purified from Trima residue (Vitalant) using Ficoll-Paque Plus and negative selection (monocyte isolation kit II (Monocyte Isolation Kit II, miltenyi Biotec) according to manufacturer's protocol by seeding 1 million CD14 + cells into a 150mm tissue culture dish (Corning) in complete RPMI growth medium supplemented with 10% human AB serum (Sigma) and 10ng/mL MCSF for 3 days, monocyte-derived macrophages (NPO) were prepared on day 3, non-adherent cells were removed and incubated with growth medium supplemented with 10% AB serum without MCSF for another 4 days using TRYPLE SELECT (Thermo FISHER SCIENTIFIC) target cells (DLD-1 or DLD-1 overexpressing SIRP- α) were labeled with CELLTRACE CFSE cell proliferation kit (Thermo FISHER SCIENTIFIC) according to manufacturer's instructions) and the corresponding ultra low cost to test of 96,000 target antibodies were incubated in a 96,000 well plate at 37 ℃ in unconjugated and conjugated anti-p- α antibody at titration 150nM to 0.2 nM; anti-SIRP-alpha-hIgG 4 antibody-CpG oligonucleotide conjugate (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO:73, conjugated to CpG oligonucleotide 7-7 (SEQ ID NO: 35)) and unconjugated anti-SIRP-alpha-hIgG 4 (heavy chain comprising the sequence of SEQ ID NO:66 and light chain comprising the sequence of SEQ ID NO: 73), anti-SIRP-alpha-hIgG 1 antibody-CpG oligonucleotide conjugates (heavy chain comprising the sequence of SEQ ID NO:68 and light chain comprising the sequence of SEQ ID NO:75, conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35)) and unconjugated anti-SIRP-alpha-hIgG 1 (heavy chain comprising the sequence of SEQ ID NO:68 and light chain comprising the sequence of SEQ ID NO: 75), and anti-SIRP-alpha-hIgG 1 AAA antibody-CpG oligonucleotide conjugates (heavy chain comprising the sequence of SEQ ID NO:67 and light chain comprising the sequence of SEQ ID NO:75, conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35)) and unconjugated anti-SIRP-alpha-hIgG 1 AAA (heavy chain comprising the sequence of SEQ ID NO:67 and light chain comprising the sequence of SEQ ID NO: 75). For flow cytometry, cells were incubated in human FcR blocking reagent (Miltenyi Biotec) and stained with fluorochrome-labeled anti-CD 33 antibodies. To eliminate macrophage/target cell adhesion from the assay, anti-CD 326 antibodies were included. In addition, a pulse geometry gate of forward scatter signal area and height is used to select single cells. Fixable viability dye (Thermo FISHER SCIENTIFIC) was used to identify living cells. Cells were obtained on FACS Canto II flow cytometer (BD Biosciences) and subsequently analyzed using FlowJo software. The percentage phagocytosis indicates the percentage of viable CD33 + macrophages that were negative for CD326 staining and positive for CFSE staining.
Results
Human monocyte-derived macrophages were incubated with SIRP-alpha overexpressing DLD-1 (FIG. 21A) or SIRP-alpha non-expressing parent DLD-1 cells (FIG. 21B) for 2 hours in the presence of various anti-SIRP-alpha antibody-CpG oligonucleotide conjugates or anti-SIRP-alpha antibodies. These results demonstrate that all tested anti-SIRP-a antibody-CpG oligonucleotide conjugates are able to promote phagocytosis of SIRP-a positive and SIRP-a negative tumor cells compared to the corresponding unconjugated anti-SIRP-a antibodies.
Example 19: antitumor Activity in mouse isogenic MC38 model containing SIRP-alpha Positive and SIRP-alpha negative tumor cells
Materials and methods
Parental MC38 colon cancer cells (ATCC) and MC38 overexpressing mouse sirpa were cultured in complete darbeck modified eagle medium (Gibco) (DMEM, 10% FBS, 1% Pen/Strep, 1% Glutmax, 1% sodium pyruvate) at 37 ℃ and 5% co 2. Cells were detached with 0.25% trypsin (Gibco) and washed twice with DMEM. Cells were resuspended in DMEM at 20E6/mL and kept on ice until use. 100uL of the suspended cells were subcutaneously implanted into the right flank of 6 week old female C57/BL6 mice (CHARLES RIVER). Tumor size was measured with a caliper one week and recorded twice after approximately 17 days after implantation starting 4 days until the duration of the study. Tumor volume was calculated using the following formula: (length x width)/2. Once the MC38 parent and MC38 SIRP-a tumors reached an average of 94mm 3 to 88mm 3, respectively, mice were randomly grouped according to tumor size and treatment started approximately 4 days post implantation. anti-SIRP- αmIgG1 conjugated to mTCpG (heavy chain comprising the sequence of SEQ ID NO:91 and light chain comprising the sequence of SEQ ID NO:111, conjugated to mouse CpG oligonucleotide 4523 (SEQ ID NO: 121)) was administered intraperitoneally at 1mg/kg every 3 days at 2 doses. Mice with tumors exceeding 2,000mm 3 or exhibiting any signs of distress were humanly sacrificed at any time during the study, according to IACUC approved animal protocols.
Results
Mice carrying SIRP- α -expressing MC38 cells (fig. 22A) or parental MC38 cells (fig. 22B) were dosed intraperitoneally (i.p.) twice, three days apart from 1mg/kg of anti-mSIRP- α antibody (square) that bound murine reactive mT-CpG or PBS control (triangle). The results demonstrate that localization of the anti-SIRP-a antibody-CpG oligonucleotide conjugate in SIRP-a positive tumor cells provides superior anti-tumor activity when compared to the parental MC38 cells that do not overexpress SIRP-a in this syngeneic tumor model when administered systemically.
Example 20: anti-SIRP-alpha antibody conjugates for anti-tumor activity in RENCA in mouse syngeneic tumor models that are difficult to treat with PD-1
Materials and methods
RENCA kidney cancer cell line (ATCC) was cultured in complete RPMI (RPMI, 10% FBS, 1 XPen/Strep, 1 XPlutamax) at 37℃and 5% CO 2. Cells were detached with trypsin 0.25% (Gibco) and washed twice with RPMI 1640 (Gibco). Cells were resuspended in RPMI 1640 at 20E6/mL and kept on ice until use. 100uL of suspended cells were subcutaneously implanted into the right flank of 6 week old female BALB/c mice (CHARLES RIVER). Tumor size was measured with a caliper one week and recorded twice after approximately 27 days after 9 days of implantation until the duration of the study. Tumor volume was calculated using the following formula: (length x width)/2. Once the tumors reached an average of 56mm 3, approximately 9 days post-implantation, mice were randomly grouped according to tumor size and treatment was started. 3 doses of anti-mSIRP-alpha conjugated to mT-CpG (heavy chain comprising the sequence of SEQ ID NO:91 and light chain comprising the sequence of SEQ ID NO:111, conjugated to mouse CpG oligonucleotide 4523 (SEQ ID NO: 121)) were administered intraperitoneally every 3 days at 10 mg/kg. In the anti-PD-1 RMP1-14 (BioXcell) treated group, once the tumor reached an average of 52mm, dosing was started on day 10 after tumor implantation. A total of 3 doses were administered at 10mg/kg 3 days apart. Mice with tumors exceeding 2,000mm 3 or exhibiting any signs of distress were humanly sacrificed at any time during the study, according to IACUC approved animal protocols.
Results
In fig. 23A, mice bearing RENCA tumor cells that were SIRP-a positive and difficult to treat with anti-PD-1 treatment were dosed intraperitoneally (i.p.) three times, three days apart from 10mg/kg of anti-mSIRP-a antibody conjugated to murine reactive mT-CpG (square) or PBS (triangle). In FIG. 23B, individual groups were dosed three times, three days apart or PBS (round) with 10mg/kg of anti-PD-1 antibody RMP1-14 (BioXcell) (triangle). These results demonstrate that anti-SIRP-a antibody-CpG oligonucleotide conjugates elicit strong single agent anti-tumor responses in this model that are difficult to treat with anti-PD-1 antibody-based single agent treatments.
Example 21: anti-tumor Activity of combination of anti-SIRP-alpha antibody conjugates with anti-PD-1 antibodies
Materials and methods
CT26 mouse colon cancer cells (ATCC) were cultured in complete RPMI (RPMI, 10% FBS, 1 XPen/Strep, 1 XPlutamax) at 37℃and 5% CO 2. Once the cells were 80% confluent, the cells were detached with trypsin 0.25% (Gibco) and washed twice with RPMI 1640 (Gibco). Cells were resuspended in RPMI 1640 at 20E6/mL and kept on ice until use. 100uL of suspended cells were subcutaneously implanted into the right flank of 6 week old female BALB/c mice (CHARLES RIVER). Tumor size was measured with a caliper one week and recorded twice after 7 days post-implantation until the duration of the study, approximately 27 days. Tumor volume was estimated using the following formula: (length x width)/2. Once the tumors reached 110mm 3, approximately 9 days post-implantation, mice were randomly grouped according to tumor size and treatment was started. The anti-mSIRP-alpha antibody-CpG oligonucleotide conjugate having a heavy chain comprising the sequence of SEQ ID NO:91 and a light chain comprising the sequence of SEQ ID NO:111 conjugated to mouse CpG oligonucleotide 4523 (SEQ ID NO: 121) was administered twice intraperitoneally at 1mg/kg 4 days apart, anti-PD-1 RMP14-1 (BioXcell) was administered twice as a single agent or in combination with the anti-mSIRP-alpha antibody-CpG oligonucleotide conjugate at 10mg/kg 4 days apart, a total of two doses apart, and anti-PD-1 twice, two days later the anti-mSIRP-alpha antibody-CpG oligonucleotide conjugate. Mice with tumors exceeding 2,000mm 3 or exhibiting any signs of distress were humanly sacrificed at any time during the study, according to IACUC approved animal protocols.
Results
Anti-tumor activity of anti-SIRP-alpha antibody conjugates administered in sub-optimal doses in combination with anti-PD-1 antibodies was tested in a mouse syngeneic tumor model. Mice bearing CT26 tumor cells were treated intraperitoneally (i.p.) with 1mg/kg of an anti-mSIRP a antibody conjugated to a murine reactive mT-CpG (heavy chain comprising the sequence of SEQ ID NO:91 and light chain comprising the sequence of SEQ ID NO:111, conjugated to mouse CpG oligonucleotide 4523 (SEQ ID NO: 121)) as a single agent resulting in robust tumor growth inhibition (FIG. 24A). In follow-up studies of the same tumor model, mice were treated with a sub-optimal dose of 0.3mg/kg of anti-mSIRP-alpha antibody-CpG oligonucleotide conjugate, 10mg/kg of anti-PD-1 antibody, a combination of both, or PBS control (FIG. 24B). The results demonstrate that the combination of an anti-SIRP-a antibody-CpG oligonucleotide conjugate and an anti-PD-1 antibody elicits an enhanced anti-tumor response in a CT26 syngeneic tumor model compared to either the anti-SIRP-a antibody-CpG oligonucleotide conjugate alone or the anti-PD-1 antibody alone. Unexpectedly, the combination of anti-SIRP-a antibody-CpG oligonucleotide with anti-PD-1 antibody provided an increased anti-tumor response even though the anti-PD-1 antibody did not show anti-tumor activity in this model when administered alone. In addition, tumor growth inhibition achieved with suboptimal doses of anti-mSIRP-alpha antibody-CpG oligonucleotide conjugate in combination with anti-PD-1 was similar to 1mg/kg of single dose anti-mSIRP-alpha antibody-CpG oligonucleotide conjugate, further indicating that the combined pathway enhanced anti-tumor response.
Example 22: anti-SIRP-alpha antibody conjugates and anti-PD-L1 combinations for anti-tumor Activity in B16F10 tumor models
Materials and methods
The B16-F10 melanoma cell line (ATCC) was cultured in complete Dalberg modified eagle medium (DMEM, 10% FBS, 1% Pen/Strep, 1% Glutamax, 1% sodium pyruvate) at 37℃and 5% CO 2. Cells were detached with 0.25% trypsin (Gibco) and washed twice with DMEM (Gibco). Cells were resuspended in DMEM at 6E6/mL and kept on ice until use. 100uL of the suspended cells were subcutaneously implanted into the right flank of 6 week old female C57BL/6 mice (CHARLES RIVER). Tumor size was measured with a caliper for one week and recorded 2 to 3 times after approximately 17 days from 7 days post-implantation until the duration of the study. Tumor volume was calculated using the following formula: (length x width)/2. Once the tumors reached an average of 55mm 3, approximately 7 days post-implantation, mice were randomly grouped according to tumor size and treatment started. The conjugate was administered intraperitoneally at 30mg/kg 2 doses every 3 days. anti-PD-L1 2 doses were administered intraperitoneally every 3 days at 10 mg/kg. Mice with tumors exceeding 2,000mm 3 or exhibiting any signs of distress were humanly sacrificed at any time during the study, according to IACUC approved animal protocols. The anti-mSIRP-alpha antibody conjugate comprises a murine reactive mT-CpG oligonucleotide (SEQ ID NO: 121) conjugated to an anti-SIRP-alpha antibody comprising a heavy chain comprising the sequence of SEQ ID NO:91 and a light chain comprising the sequence of SEQ ID NO: 111. The anti-PD-L1 antibodies used were internally generated and the heavy and light chain sequences correspond to SEQ ID NOS 116 and 117, respectively.
Anti-PD-L1 heavy chain
Anti-PD-L1 light chain
Results
The anti-tumor activity of the combination of an anti-SIRP-a antibody conjugate with an anti-PD-L1 antibody was tested in a mouse syngeneic tumor model. Mice bearing B16F10 tumor cells were treated intraperitoneally (i.p.) with 30mg/kg of anti-SIRP-a antibody conjugated to murine reactive mT-CpG 4523, 10mg/kg of anti-PD-L1 antibody, a combination of both, or PBS control (fig. 25). The results demonstrate that the combination of an anti-SIRP-a antibody-CpG oligonucleotide conjugate and an anti-PD-L1 antibody elicits an enhanced anti-tumor response in a B16F10 syngeneic tumor model compared to either the anti-SIRP-a antibody-CpG oligonucleotide conjugate alone or the anti-PDL-1 antibody alone.
In summary, anti-SIRP-a antibody-CpG oligonucleotide conjugates were observed to specifically target bone marrow cells and trigger TLR9 signaling (e.g., via SIRP-a and fcγr junctions), leading to robust cell activation and cytokine induction in cultured PBMCs. The anti-SIRP-alpha antibody-CpG oligonucleotide conjugate enhances activation of bone marrow cells in the presence of SIRP-alpha expressing tumor cells and promotes phagocytosis of tumor cells independent of SIRP-alpha expression. Localization of anti-mSIRP-alpha antibody-mouse reactive CpG oligonucleotide conjugates to SIRP-alpha positive tumors demonstrated robust and curative single agent activity in multiple mouse models, including tumor types that are difficult to treat with anti-PD-1 treatment. In an isogenic tumor model, anti-mSIRP-alpha antibody-mouse reactive CpG oligonucleotide conjugates in combination with anti-PD-1 enhance tumor regression.
Example 23: anti-SIRP-alpha antibody conjugates inhibit tumor growth in CT26 tumor bearing mice that are non-responsive to prior anti-PD-1 therapies
Materials and methods
CT26 colon cancer cell line (ATCC) was cultured in complete Rosweil Parker 1640 medium supplemented with 10% FBS (Gibco), 100U/mL penicillin and 100ug/mL streptomycin (Gibco) and 2mM GlutaMAX (Gibco) RPMI-1640 at 37℃and 5% CO 2. Cells were detached with 0.25% trypsin (Gibco) and washed twice with RPMI-1640 (Gibco). Cells were resuspended in RPMI-1640 at 20 6 cells/mL and kept on ice until use. 100uL of the cell suspension, 2E6 CT26 cells were subcutaneously implanted into the right flank of 6 week old female BALB/C mice (CHARLES RIVER). Tumor size was measured with a caliper for one week and recorded 2 to 3 times after approximately 26 days from 6 days post-implantation until the duration of the study. Tumor volume was calculated using the following formula: (length x width)/2.
Once the tumors reached an average of 108mm 3, mice were randomized according to tumor size on day 6 post-implantation and started with PBS or anti-PD-1 RMP1-14 (BioXcell) (10 mg/kg). The treatment was performed twice intraperitoneally 3 days apart. Tumor volumes were measured two days after the second anti-PD-1 dose. If the tumor measurement exceeds the initial size and is greater than 250mm 3, the anti-PD-1 treated mice are considered non-responders. Mice that were unresponsive to anti-PD-1 were re-randomized into 3 new treatment groups according to average tumor volume of 338mm 3 on day 11. Day 11 mice received 1mg/kg of anti-SIRP-alpha antibody conjugate alone (group 1) or in combination with 10mg/kg of anti-PD-1 (group 2), or continued to receive 10mg/kg of anti-PD 1 monotherapy (group 3). Treatments were administered intraperitoneally every 3 days at a total of 2 doses. Mice with tumors exceeding 2,000mm 3 or exhibiting any signs of distress were humanly sacrificed at any time during the study, according to IACUC approved animal protocols. The anti-mSIRP-alpha antibody conjugate comprises a murine reactive mT-CpG oligonucleotide (SEQ ID NO: 121) conjugated to an anti-SIRP-alpha antibody comprising a heavy chain comprising the sequence of SEQ ID NO:91 and a light chain comprising the sequence of SEQ ID NO: 111.
Results
The activity of the anti-SIRP-a antibody-CpG oligonucleotide conjugates in this CT26 tumor model was tested in mice that were non-responsive to prior anti-PD-1 treatment as described above. As shown in fig. 26, the anti-SIRP-a antibody-CpG oligonucleotide conjugate showed robust tumor growth inhibition at 1mg/kg in CT26 tumor-bearing mice that were non-responsive to prior anti-PD-1 treatment. Tumor eradication was observed in all 9 mice treated with 1mg/kg anti-SIRP-alpha antibody conjugate alone. Treatment with another 2 doses of 10mg/kg anti-PD-1 monotherapy continued only slightly delayed tumor growth compared to the PBS control group. The combination of 10mg/kg anti-PD-1 with 1mg/kg anti-SIRP-a antibody conjugate resulted in eradication of 9 tumors in 10 mice and did not appear to further enhance tumor growth inhibition compared to 1mg/kg anti-SIRP-a antibody conjugate treatment alone. These results demonstrate that treatment with anti-SIRP-a antibody-CpG oligonucleotide conjugates has strong anti-tumor activity even against tumors that were previously unresponsive to anti-PD-1 treatment.
Example 24: target binding characteristics of anti-SIRP-alpha antibody conjugates
Materials and methods
Binding of anti-SIRP-a antibody conjugates was measured using a Biacore 8K high-throughput, high-sensitivity SPR system (Cytiva, global LIFE SCIENCES Solutions USA LLC, marlborough, MA) equipped with a class S sensor chip. The interaction of the conjugate with SIRP-a was analyzed by flowing His-tagged SIRP-a over protein a captured anti-SIRP-a antibody conjugate on a Biacore series S protein a sensor chip. The anti-SIRP-alpha antibody portion of the conjugate comprises a heavy chain comprising the sequence of SEQ ID NO:66 and a light chain comprising the sequence of SEQ ID NO:73, conjugated to CpG oligonucleotides 7-7 (SEQ ID NO: 35). The SIRP-alpha analytes used for the determination are shown in Table 18.
Table 18. Sirp-a analytes.
Rat sirpa-ECD:
All SIRP-alpha analytes were injected in a single cycle kinetic mode at a nominal starting concentration of 100nM at 3-fold serial dilutions, except for rat SIRP-alpha. The higher initial concentration of 300nM was used to assess binding to rat SIRP-alpha at pH 7.4. The association time was monitored for 120s and the dissociation time was monitored for 600s at a flow rate of 30. Mu.L/min. The surface was regenerated with two 15s pulses of 75mM phosphoric acid at pH 1.65 at a flow rate of 30. Mu.L/min. Experiments were performed at 25 ℃ or 37 ℃ and at pH 6.0 or 7.4. The assay buffer was 1 XPBS 0.01% Tween-20 at pH 6.0 and 1 XHBS-EP+ (10mM HEPES,pH7.4, 150mM NaCl,3mM EDTA,0.05% (v/v) surfactant P20) at pH 7.4. All data points were collected in duplicate or more times.
Data were processed and analyzed using Biacore 8K evaluation software 4.0.8.20368 edition (Cytiva, global LIFE SCIENCES Solutions USA LLC, marlborough, MA). Subtracting the reference reaction of flow cell 1 from the active reaction of flow cell 2, resulting in subtraction data (2-1). Next, the response from the most recent buffer blank injection was subtracted from the reference subtracted data (2-1) to obtain double reference data. These double reference data were fitted to a simple 1:1langmuir binding model with mass transport to determine apparent association (ka) and dissociation rate constants (kd). The apparent equilibrium dissociation constant or affinity constant is then calculated based on its ratio, e.g. (K D =kd/ka).
Results
The results of the binding assays are shown in table 19. The results indicate that the conjugate shows higher binding affinity for SIRP-a for all test species at both temperatures at lower pH. Without wishing to be bound by theory, it is believed that these features confer advantageous binding properties to the anti-SIRP-a antibody conjugate, as the acidic microenvironment of the tumor (pH 5.6 to pH 6.8) is a marker of malignant tumor cells.
TABLE 19K D measurement of anti-SIRP-alpha antibody conjugates
Example 25: efficacy of anti-SIRP-alpha antibody conjugates to induce TLR9 pathway signaling
Materials and methods
The THP-1-bis hTLR9 cell line (thpd-nfis, invitrogen) was derived from human THP-1 monocytes engineered to overexpress the human TLR9 (hTLR 9) gene and having two reporter genes, allowing for simultaneous assessment of the NF-kB pathway by monitoring the activity of inducible Secreted Embryonic Alkaline Phosphatase (SEAP) and the Interferon Regulatory Factor (IRF) pathway by monitoring the activity of inducible secreted Lucia luciferase. Briefly, 250,000 cells were plated in 96-well flat bottom plates (Nunc) in 100uL growth medium (RPMI 1640 (Gibco), 2mM L-glutamine (Gibco), 25mM HEPES (Gibco), 10% FBS (Gibco), 100U/mL penicillin (Gibco), 100ug/mL streptomycin (Gibco), 100ug/mL Normacin (Invivogen), 8 steps of 2-fold serial dilutions of an anti-SIRP-alpha antibody or an anti-SIRP-alpha antibody conjugate were added to 50uL cells at a final concentration ranging from 200nM to 1.56nM, 10ug/mL F (ab') 2 fragment goat anti-human, igG Fc gamma fragment specificity (Jackson Immuno Research) were added as cross-linker to 50uL growth medium, cells were incubated at 37℃for 24 hours from the supernatant using three parameters of three-parameter regression in sm9 (GraPad) to induce the light-protein) and the light chain comprising a linear extension of the anti-SIRP-alpha antibody sequence of SEQ ID strand of SEQ ID of 35 to 35, and the anti-SIRP-antibody sequence comprising the light chain of SEQ ID 35.
Results
FIG. 27A demonstrates that an anti-SIRP-alpha antibody, cpG oligonucleotide conjugate, elicits 10-fold stronger IRF induction than T-CpG with 84nM EC 50 and 8.5nM EC 50. Similarly, anti-SIRP-alpha antibody conjugates induce NF-kB, EC 50 10.5.5 nM, approximately 6-fold higher than the free CpG presented in FIG. 27B. Unconjugated anti-SIRP-alpha antibodies do not elicit a response.
Overall, these data demonstrate that enhanced TLR9 engagement stabilization in response to T-CpG by direct targeting of anti-SIRP-a antibody conjugates induces NF-kB and IRF pathways.

Claims (150)

1. A conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment is linked to one or more Q tag peptides (Q) comprising at least one glutamine residue, and wherein each immunomodulatory oligonucleotide is linked to a Q tag peptide via a amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
Wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61);
Wherein each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of
* And/>* Indicates the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection to the joint L;
each T 1 is independently O or S;
Each T 2 is S-;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/># Indicates the point of attachment to the rest of the oligonucleotide;
Z is O or S;
U 5' is-H or halogen;
r 5' is-H or methoxy;
Rc 1 is-H or methoxy;
Rg 1、Rg2、Rg3 and Rg 4 are H;
r 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer from 0 to 2.
2. A conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-alpha polypeptide and (ii) one or more immunomodulatory oligonucleotides (P),
Wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising the amino acid sequence of RPQGF (SEQ ID NO: 47);
Wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61);
And wherein each immunomodulatory oligonucleotide (P) is linked to a Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof.
3. A conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof
Each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of * And/>* Indicates the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection with the joint L.
4. A conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof
Each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of * And/>* Indicates the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection with the joint L.
5. A conjugate comprising an antibody or antigen binding fragment (Ab) thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide, wherein the antibody comprises: two antibody light chains each comprising a light chain Variable (VL) domain comprising a CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), a CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and a CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); two antibody heavy chains each comprising a heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57), and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58); and two Q tag peptides comprising peptide sequence RPQGF (SEQ ID NO: 47); wherein the Q tag peptides are each linked to the C-terminus of one of the antibody heavy chains; and wherein at least one of the Q tag peptides is linked to an immunomodulatory oligonucleotide (P) via an amide bond and a linker (L) to the glutamine residue of the Q tag peptide, as shown in fig. 9A or 9B.
6. A conjugate comprising an antibody (Ab) that specifically binds to an extracellular domain of a human SIRP-a polypeptide, at least one Q tag peptide sequence comprising a glutamine residue, and at least one immunomodulatory oligonucleotide (P), wherein the antibody or antigen-binding fragment thereof (Ab) comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); wherein the Q tag peptide sequence is naturally occurring or synthetic; wherein each immunomodulatory oligonucleotide is linked to a Q tag via an amide bond to the glutamine residue and a linker (L); and wherein at least one Q tag peptide sequence is selected from the group consisting of SEQ ID NOS: 39-55.
7. The conjugate of any one of claims 1-6, wherein the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAP GKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62), and wherein the VL domain comprises the amino acid sequence of SYELTQPPSVSVSP GQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPE RFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTQ LTVL(SEQ ID NO:63).
8. The conjugate of any one of claims 1-6, wherein the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAP GKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62), and wherein the VL domain comprises the amino acid sequence of SYELTQPPSVSV SPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIP ERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGT KLTVL(SEQ ID NO:64).
9. The conjugate of any one of claims 1-6, wherein the VH domain comprises the amino acid sequence of EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAP GKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARETWNHLFDYWGQGTLVTVSS(SEQ ID NO:62), and wherein the VL domain comprises the amino acid sequence of SYELTQPPSVSVSP GQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPE RFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFGGGTE LTVL(SEQ ID NO:65).
10. The conjugate of any one of claims 1-9, wherein the antibody is a monoclonal antibody.
11. The conjugate of any one of claims 1-10, wherein the antibody is a Fab, F (ab ') 2, fab' -SH, fv, or scFv antibody or antibody fragment.
12. The conjugate of any one of claims 1-11, wherein the antibody is a humanized antibody, a human antibody, or a chimeric antibody or fragment thereof.
13. The conjugate of any one of claims 1-12, wherein the antibody comprises an antibody heavy chain comprising the VH domain and Fc region.
14. The conjugate of claim 13, wherein the antibody heavy chain comprises a human IgG1, human IgG2, or human IgG4 Fc region.
15. The conjugate of claim 13, wherein the antibody heavy chain comprises:
(a) A human IgG1 Fc region comprising an L234A, L a and/or G237A substitution, the amino acid positions being numbered according to the EU index;
(b) A wild-type human IgG1 Fc region;
(c) A human IgG1 Fc region comprising an N297A substitution, amino acid positions numbered according to the EU index;
(d) A human IgG1 Fc region comprising a D265A substitution, the amino acid positions being numbered according to the EU index;
(e) A wild-type human IgG2 Fc region;
(f) A human IgG2 Fc region comprising an N297A substitution, amino acid positions numbered according to the EU index; or (b)
(G) Comprising an S228P substituted human IgG4 Fc region, the amino acid positions being numbered according to the EU index.
16. The conjugate of any one of claims 1-13, wherein the antibody comprises an antibody heavy chain comprising an amino acid sequence having a C-terminal Q tag peptide EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGRPQGFGPP(SEQ ID NO:66).
17. The conjugate of any one of claims 1-13, wherein the antibody comprises an antibody heavy chain comprising an amino acid sequence having a C-terminal Q tag peptide EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP(SEQ ID NO:67).
18. The conjugate of any one of claims 1-13, wherein the antibody comprises an antibody heavy chain comprising an amino acid sequence having a C-terminal Q tag peptide EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP(SEQ ID NO:68).
19. The conjugate of any one of claims 1-13, wherein the antibody comprises an antibody heavy chain comprising amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:88).
20. The conjugate of any one of claims 1-13, wherein the antibody comprises an antibody heavy chain comprising amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:87).
21. The conjugate of any one of claims 1-13, wherein the antibody comprises an antibody heavy chain comprising amino acid sequence EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWNHLFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:89).
22. The conjugate of claim 13, wherein the Fc region comprises an N297A substitution, amino acid positions numbered according to the EU index.
23. The conjugate of claim 22, wherein the conjugate further comprises an immunomodulatory oligonucleotide P linked to Q295 of the Fc region residue, as shown in the formula: wherein L is a linker moiety linked to Q295 of the Fc region via an amide bond.
24. The conjugate of any one of claims 1-23, wherein the antibody comprises an antibody light chain comprising the VL domain and a light chain Constant (CL) domain, and wherein the CL domain comprises an amino acid sequence GQPKANPTVTLFPPSSEELQANK ATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYA ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:69).
25. The conjugate of any one of claims 1-23, wherein the antibody comprises an antibody light chain comprising the VL domain and a light chain Constant (CL) domain, and wherein the CL domain comprises an amino acid sequence GQPKANPTVTLFPPSSEELQANKA TLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSSDKYAA SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO:70).
26. The conjugate of any one of claims 1-23, wherein the antibody comprises an antibody light chain comprising the VL domain and a light chain Constant (CL) domain comprising an amino acid sequence GQPKAAPSVTLFPPSSEELQANKA TLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA SSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ IDNO:71).
27. The conjugate of any one of claims 1-23, wherein the antibody comprises an antibody light chain comprising an amino acid sequence selected from the group consisting of SEQ ID nos 72-80.
28. The conjugate of any one of claims 1-6, wherein the antibody comprises:
(a) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79;
(b) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79;
(c) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 79;
(d) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75;
(e) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75;
(f) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 75;
(g) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 68 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73;
(h) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 67 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73; or (b)
(I) An antibody heavy chain comprising the amino acid sequence of SEQ ID NO. 66 having a C-terminal Q tag peptide and an antibody light chain comprising the amino acid sequence of SEQ ID NO. 73.
29. The conjugate of any one of claims 1-15 and 19-27, wherein the one or more Q tag peptides (Q) each comprise a peptide sequence having 5 to 15 amino acid residues.
30. The conjugate of any one of claims 1, 6-15 and 19-29, wherein each of the one or more Q tag peptides is naturally occurring.
31. The conjugate of claim 29, wherein the one or more Q tag peptides each comprise a sequence independently selected from the group consisting of SEQ ID NOs 39-55.
32. The conjugate of claim 29, wherein the one or more Q tag peptides each comprise peptide sequence RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48) or RPQGFGPP (SEQ ID NO: 49).
33. The conjugate of claim 29, wherein each of the one or more Q tag peptides comprises peptide sequence RPQGF (SEQ ID NO: 47).
34. The conjugate of claim 29, wherein the at least one Q tag peptide sequence comprises peptide sequence RPQGF (SEQ ID NO: 47), RPQGFPP (SEQ ID NO: 48) or RPQGFGPP (SEQ ID NO: 49).
35. The conjugate of claim 29, wherein the at least one Q tag peptide sequence comprises peptide sequence RPQGFGPP (SEQ ID NO: 49).
36. The conjugate of any one of claims 1-4 and 6-35, wherein the antibody comprises two antibody heavy chains and two antibody light chains, and wherein one or both heavy chains further comprise the Q tag.
37. The conjugate of any one of claims 1-4 and 6-35, wherein the Q tag is fused to the C-terminus of one or both of the heavy chains.
38. The conjugate of any one of claims 1-4, 6-15, and 19-35, wherein the Q tag is within the Fc domain.
39. The conjugate of any one of claims 1-4 and 6-35, wherein the antibody comprises two antibody heavy chains and two antibody light chains, and wherein one or both light chains further comprise the Q tag.
40. The conjugate of any one of claims 1-39, wherein the conjugate induces activation of TLR 9.
41. The conjugate of any one of claims 1-4 and 6-35, wherein 1 or 2Q tags are attached to the antibody.
42. The conjugate of any one of claims 1-41, wherein the DAR of the conjugate is 1 or 2.
43. The conjugate of any one of claims 1-42, wherein the linker L comprises a polyethylene glycol moiety.
44. The conjugate of any one of claims 1-2 and 5-43, wherein the linker L is
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to T 3, and/>Indicating the point of attachment to the remainder of the conjugate.
45. The conjugate of any one of claims 1-2 and 5-44, wherein Z is S.
46. The conjugate of any one of claims 1-2 and 5-45, wherein the oligonucleotide P comprises at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S-.
47. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 1.
48. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 2.
49. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 3.
50. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 5.
51. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 6.
52. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 7.
53. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 8.
54. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 9.
55. The conjugate of claim 46, wherein the at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 10.
56. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 11.
57. The conjugate of claim 46, wherein the at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 12.
58. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 13.
59. The conjugate of claim 46, wherein the at least one pair of gem T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 14.
60. The conjugate of claim 46, wherein the at least one pair of gems T 1 and T 2, wherein T 1 is S and T 2 is S, is located at the 3' position of nucleoside residue 15.
61. The conjugate of any one of claims 1-2 and 5-60, wherein the oligonucleotide P comprises at least two pairs of gems T 1 and T 2, wherein T 1 is S and T 2 is S-.
62. The conjugate of any one of claims 1-2 and 5-61, wherein R 5' is H.
63. The conjugate of any one of claims 1-2 and 5-61, wherein R 5' is methoxy.
64. The conjugate of any one of claims 1-2 and 5-61, wherein Rc 1 is H.
65. The conjugate of any one of claims 1-2 and 5-63, wherein Rc 1 is methoxy.
66. The conjugate of any one of claims 1-2 and 5-65, wherein R 2 is methyl.
67. The conjugate of any one of claims 1-2 and 5-65, wherein R 2 is H.
68. The conjugate of any one of claims 1-2 and 5-67, wherein U 5' is bromo.
69. The conjugate of any one of claims 1-2 and 5-67, wherein U 5' is-H.
70. The conjugate of any one of claims 1-69, wherein m is an integer from 20 to 25.
71. The conjugate of claim 70, wherein m is 24.
72. The conjugate of any one of claims 1-2 and 5-45, wherein each P independently comprises an oligonucleotide selected from table 2, table 14 and table 15.
73. The conjugate of any one of claims 1-2 and 5-45, wherein each linker (L) and each immunomodulatory oligonucleotide (P) independently comprises an oligonucleotide selected from table 2, table 16 and table 17.
74. The conjugate of any one of claims 1-2 and 5-45, wherein each P independently comprises an oligonucleotide selected from the group consisting of SEQ ID NOs 1-38 and 129-166.
75. The conjugate of any one of claims 1-2 and 5-45, wherein each immunomodulatory oligonucleotide P is independently
Wherein the method comprises the steps of
B and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5;
* Indicating the point of attachment of the immunomodulatory oligonucleotide P to the remainder of the conjugate;
x 5' is a containing structure A 5' terminal nucleoside of (2);
X 3' is a containing structure 3' Terminal nucleoside of (2);
y PTE is a containing structure Wherein is indicates the point of attachment to the rest of the oligonucleotide, and/>Indicating the point of attachment to the linker L, or if L is not present,/>Indicating a point of attachment to the Q tag peptide via an amide bond at the glutamine residue; /(I)
Y 3' is a containing structureIs a terminal phosphotriester of (a);
each X N is independently an inclusion structure A nucleoside of (2);
Each Y N is independently an inclusion structure Is a nucleoside linker; wherein each B N is independently a modified or unmodified nucleobase;
Each R N is independently-H or-O-C 1-4 -alkyl, wherein said C 1-4 -alkyl of said-O-C 1-4 -alkyl is optionally further substituted with-O-C 1-C4 -alkyl;
B 5 'and B 3' are independently modified or unmodified nucleobases;
R 5 'and R 3' are independently-H or-O-C 1-C4 -alkyl, wherein the C 1-4 -alkyl of the-O-C 1-4 -alkyl is optionally further substituted by-O-C 1-4 -alkyl;
each T 1 is independently O or S;
Each T 2 is independently O-or S-; and
T 3 is a group comprising an oligoethylene glycol moiety; and
R 1 is C 1-4 -alkylene-hydroxy.
76. The conjugate of claim 75, wherein b is 3.
77. The conjugate of claim 75 or claim 76, wherein:
(i) P comprises at least one modified nucleoside X N;
(ii) P comprises at least one modified internucleoside linker Y N, wherein at least one of T 1 or T 2 is S; or (b)
(Iii) Both (i) and (ii).
78. The conjugate of any one of claims 75-77, wherein P comprises at least one dithiophosphate or phosphorothioate internucleoside linker.
79. The conjugate of any one of claims 75-78, wherein P comprises 0, 1, 2, or 3 phosphorodithioate internucleoside linkers.
80. The conjugate of any one of claims 75-79, wherein P comprises a modified nucleoside selected from the group consisting of: 2' -O-alkyl nucleosides, 2' -O-alkoxyalkyl nucleosides, 2' -deoxynucleosides, and ribonucleosides.
81. The conjugate of claim 80, wherein the modified nucleoside is selected from the group consisting of: 5-bromo-2 '-O-methyluridine, 5-bromo-2' -deoxyuridine, 2 '-O-methyluridine, 2' -deoxyuridine, 2 '-O-methylthymidine, 2' -O-methylcytidine, 2'-O- (2-methoxyethyl) thymidine and 8-oxo-7, 8-dihydro-2' -deoxyguanosine.
82. The conjugate of any one of claims 75-81, wherein X 5 ' is 5-bromo-2 ' -O-methyluridine, 5-bromo-2 ' -deoxyuridine, 2' -O-methyluridine, or 2' -deoxyuridine.
83. The conjugate of any one of claims 75-82, wherein Y 3 ' or the Y N at the 3' position of X 5 ' comprises an unsubstituted or substituted phosphorothioate.
84. The conjugate of any one of claims 75-83, wherein Y PTE is:
Wherein Z is O or S; d is an integer from 0 to 95; two on the right side of the structure * Indicates the point of attachment to the adjacent nucleoside X N in the oligonucleotide P, and to the left of the structure/>Indicating the point of connection with the joint L.
85. The conjugate of any one of claims 75-83, wherein Y PTE is:
Wherein Z is O or S; d is an integer from 0 to 95; two on the right side of the structure * Indicates the point of attachment to the adjacent nucleoside X N in the oligonucleotide P, and one/>, to the left of the structureIndicating the point of connection with the joint L.
86. The conjugate of claim 84 or 85, wherein Z of the Y PTE is S.
87. The conjugate of any one of claims 84-86, wherein d is an integer from 1 to 25.
88. The conjugate of any one of claims 75-87, wherein the linker L comprises a polyethylene glycol moiety.
89. The conjugate of any one of claims 75-88, wherein the linker L is
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to Y PTE, and/>Indicating the point of attachment to the remainder of the conjugate.
90. The conjugate of any one of claims 1-2 and 5-89, wherein P comprises one or more CpG sites.
91. The conjugate of any one of claims 1-2 and 5-90, wherein P comprises at least 3 CpG sites.
92. The conjugate of any one of claims 1-4 and 6-91, wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the Q tag peptides are each linked to the C-terminus of one of the antibody heavy chains; and wherein one of the Q tag peptides is linked to an immunomodulatory oligonucleotide (P) via an amide bond to a glutamine residue of the Q tag peptide and a linker (L).
93. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
94. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
95. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
96. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
97. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
98. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
99. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
100. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
101. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
102. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
103. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
104. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
105. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
106. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
107. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 75; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
108. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
109. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
110. A conjugate comprising an antibody (Ab) and an immunomodulatory oligonucleotide (P), wherein the antibody comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
111. The conjugate of any one of claims 93-110, wherein the DAR for the conjugate is 1.
112. The conjugate of any one of claims 93-110, wherein the DAR of the conjugate is 2.
113. A method for preparing a conjugate comprising (i) an antibody or antigen binding fragment thereof (Ab) that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) an immunomodulatory oligonucleotide (P), the method comprising:
Contacting the Ab with the oligonucleotide P in the presence of transglutaminase;
wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47);
Wherein each P independently comprises the formula:
Wherein the method comprises the steps of
X 5 'is a 5' terminal nucleoside;
X 3 'is a 3' terminal nucleoside;
y PTE is an internucleoside phosphotriester;
Y 3' is a terminal phosphotriester;
Each X N is independently a nucleoside;
each Y N is independently an internucleoside linker;
b and c are each independently integers from 1 to 25; provided that the sum of b and c is at least 5; and
L is a linker moiety comprising a terminal amine; and
Wherein the antibody or antigen binding fragment comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61).
114. The method of claim 113, wherein each immunomodulatory oligonucleotide is independently an oligonucleotide of formula (C) or formula (D) selected from the group consisting of the oligonucleotides of table 15 and table 17.
115. The method of claim 113 or claim 114, wherein each Q tag peptide sequence comprises peptide sequence RPQGFGPP (SEQ ID NO: 49).
116. The method of any one of claims 113-115, wherein the Ab comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides (Q) having at least one glutamine residue; one Q tag peptide is attached to the C-terminus of each of the two antibody heavy chains.
117. The method of any one of claims 113-116, wherein the DAR of the conjugate is 1 or 2.
118. The method of claim 117, wherein the DAR of the conjugate is 1, and the method further comprises isolating the conjugate with DAR 1 from free oligonucleotide, unconjugated antibody, and a conjugate with DAR 2.
119. A pharmaceutical composition comprising the conjugate of any one of claims 1-112 and a pharmaceutically acceptable carrier.
120. A method for treating cancer, the method comprising administering to an individual an effective amount of the conjugate of any one of claims 1-112 or the pharmaceutical composition of claim 119.
121. The method of claim 120, wherein the cancer is lung cancer, intrahepatic bile duct cancer, squamous cell carcinoma, brain tumor, glioblastoma, head and neck cancer, hepatocellular carcinoma, colorectal cancer, skin cancer, lung cancer, endometrial cancer, liver cancer, bladder cancer, gastric or gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, urinary tract cancer, urothelial cancer, breast cancer, peritoneal cancer, uterine cancer, salivary gland cancer, renal or renal cancer, prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer or testicular cancer, melanoma, multiple myeloma and B-cell lymphoma, non-hodgkin's lymphoma (NHL), acute Lymphoblastic Leukemia (ALL), chronic Lymphoblastic Leukemia (CLL), acute Myelogenous Leukemia (AML), merkel cell cancer, hairy cell leukemia, or Chronic Myelogenous Leukemia (CML), including metastases thereof.
122. The method of claim 121, wherein the cancer is melanoma or renal cancer.
123. The method of any one of claims 120-122, wherein the cancer is predicted to be non-responsive to PD-L1 or a PD-1 inhibitor.
124. The method of any one of claims 120-122, wherein the individual does not achieve a significant therapeutic response to PD-L1 or a PD-1 inhibitor.
125. The method of any one of claims 120-124, wherein the individual has been treated with PD-L1 or a PD-1 inhibitor prior to administration of the conjugate or composition.
126. The method of any one of claims 120-125, wherein the cells of the cancer express human SIRP-a.
127. The method of any one of claims 120-125, wherein the cells of the cancer do not express human SIRP-a.
128. The method of any one of claims 120-127, further comprising administering to the individual an additional therapeutic agent.
129. The method of claim 128, wherein the additional therapeutic agent comprises an immunotherapy, chemotherapy, radiation therapy, cell-based therapy, an anti-cancer vaccine, or an anti-cancer agent.
130. The method of any one of claims 120-127, further comprising administering to the individual PD-L1 or a PD-1 inhibitor.
131. The method of claim 130, wherein the PD-L1 or PD-1 inhibitor is an antibody that binds PD-L1 or PD-1.
132. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
133. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
134. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 35; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
135. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
136. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
137. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID No. 163; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
138. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 68; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
139. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 67; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
140. The method of any one of claims 120-131, wherein the antibody of the conjugate comprises two antibody light chains, two antibody heavy chains, and two Q tag peptides; wherein the antibody light chains each comprise the amino acid sequence of SEQ ID NO. 73; wherein the antibody heavy chains are each linked to a C-terminal Q tag peptide (Q) and comprise the amino acid sequence with Q of SEQ ID No. 66; wherein said immunomodulatory oligonucleotide and said linker comprise the structure of SEQ ID NO. 34; and wherein at least one of the two Q tag peptides is linked to the immunomodulatory oligonucleotide via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide.
141. A method for activating bone marrow cells, the method comprising administering to an individual in need thereof an effective amount of the conjugate of any one of claims 1-112 or the pharmaceutical composition of claim 119.
142. A method for inducing TLR9 signalling in a bone marrow cell, the method comprising administering to an individual in need thereof an effective amount of the conjugate of any one of claims 1-112 or the pharmaceutical composition of claim 119.
143. A kit or article of manufacture comprising a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P), wherein the antibody or antigen-binding fragment is linked to one or more Q tag peptides (Q) comprising at least one glutamine residue, and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to a glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof;
Wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61);
Wherein each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of
* And/>* Indicates the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection to the joint L; /(I)
Each T 1 is independently O or S;
Each T 2 is S-;
T 3 is a group Wherein/>Indicates the point of attachment to L, and wherein/># Indicates the point of attachment to the rest of the oligonucleotide;
Z is O or S;
U 5' is-H or halogen;
r 5' is-H or methoxy;
Rc 1 is-H or methoxy;
Rg 1、Rg2、Rg3 and Rg 4 are H;
r 3' is methoxy;
R 1 is- (CH 2)3 -OH;
r 2 is-H or methyl; and
N is an integer from 0 to 2.
144. A kit or article of manufacture comprising a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-alpha polypeptide and (ii) one or more immunomodulatory oligonucleotides (P),
Wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q) comprising amino acid sequence RPQGF (SEQ ID NO: 47);
Wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61);
And wherein each immunomodulatory oligonucleotide (P) is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
Wherein the method comprises the steps of Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof.
145. A kit or article of manufacture comprising a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating the point of attachment of each Q to the antibody or antigen binding fragment (Ab) thereof
Each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47);
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of * And/>* Indicates the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection with the joint L. /(I)
146. A kit or article of manufacture comprising a conjugate comprising (i) an antibody or antigen-binding fragment thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide and (ii) one or more immunomodulatory oligonucleotides (P); wherein the antibody or antigen binding fragment is linked to one or more Q tag peptides (Q); wherein the antibody or antigen binding fragment (Ab) thereof comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYY PASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); and wherein each immunomodulatory oligonucleotide is linked to the Q tag peptide via an amide bond to the glutamine residue of the Q tag peptide and a linker (L), as shown in formula (a):
wherein:
Indicating that each Q independently comprises a Q tag peptide sequence RPQGF (SEQ ID NO: 47) at the point of attachment of each Q to the antibody or antigen-binding fragment (Ab) thereof;
Each L is independently a bond or linker moiety
Wherein m is an integer in the range of about 0 to about 50, and whereinIndicates the point of attachment to P, and/>Indicating a point of attachment to the remainder of the conjugate linked to Q via an amide bond with the glutamine residue; and
Each P is independently an immunomodulatory oligonucleotide comprising the structure:
Wherein the method comprises the steps of * And/>* Indicates the point of attachment within the oligonucleotide, and wherein/>Indicating the point of connection with the joint L.
147. A kit or article of manufacture comprising a conjugate comprising an antibody or antigen binding fragment (Ab) thereof that specifically binds to an extracellular domain of a human SIRP-a polypeptide, wherein the antibody comprises: two antibody light chains each comprising a light chain Variable (VL) domain comprising a CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), a CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and a CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); two antibody heavy chains each comprising a heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57), and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58); and two Q tag peptides comprising peptide sequence RPQGF (SEQ ID NO: 47); wherein the Q tag peptides are each linked to the C-terminus of one of the antibody heavy chains; and wherein at least one of the Q tag peptides is linked to an immunomodulatory oligonucleotide (P) via an amide bond and a linker (L) to a glutamine residue of the Q tag peptide, as shown in fig. 9A or 9B.
148. A kit or article of manufacture comprising a conjugate comprising an antibody (Ab) that specifically binds to an extracellular domain of a human SIRP-a polypeptide, at least one Q tag peptide sequence comprising a glutamine residue, and at least one immunomodulatory oligonucleotide (P), wherein the antibody or antigen-binding fragment thereof (Ab) comprises: (a) A heavy chain Variable (VH) domain comprising CDR-H1 comprising the amino acid sequence of SNAMS (SEQ ID NO: 56), CDR-H2 comprising the amino acid sequence of GISAGGSDTYYPASVKG (SEQ ID NO: 57) and CDR-H3 comprising the amino acid sequence of ETWNHLFDY (SEQ ID NO: 58), and (b) a light chain Variable (VL) domain comprising CDR-L1 comprising the amino acid sequence of SGGSYSSYYYA (SEQ ID NO: 59), CDR-L2 comprising the amino acid sequence of SDDKRPS (SEQ ID NO: 60) and CDR-L3 comprising the amino acid sequence of GGYDQSSYTNP (SEQ ID NO: 61); wherein the Q tag peptide sequence is naturally occurring or synthetic; wherein each immunomodulatory oligonucleotide is linked to a Q tag via an amide bond to the glutamine residue and a linker (L); and wherein at least one Q tag peptide sequence is selected from the group consisting of SEQ ID NOS: 39-55.
149. The kit or article of manufacture of any of claims 141-148, further comprising instructions for using the conjugate to treat cancer.
150. The kit or article of manufacture of any one of claims 141-149, wherein the Q tag peptide sequence comprises peptide sequence RPQGFGPP (SEQ ID NO: 49).
CN202280067118.9A 2021-08-25 2022-08-24 SIRP-alpha antibodies and conjugates Pending CN118043078A (en)

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US202163256270P 2021-10-15 2021-10-15
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PCT/US2022/075381 WO2023028511A1 (en) 2021-08-25 2022-08-24 Sirp-alpha antibodies and conjugates

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