GB2552041A - Compositions of antibody construct-agonist conjugates and methods thereof - Google Patents

Compositions of antibody construct-agonist conjugates and methods thereof Download PDF

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Publication number
GB2552041A
GB2552041A GB1620828.2A GB201620828A GB2552041A GB 2552041 A GB2552041 A GB 2552041A GB 201620828 A GB201620828 A GB 201620828A GB 2552041 A GB2552041 A GB 2552041A
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Prior art keywords
compound
antibody
conjugate
salt
seq
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GB1620828.2A
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GB201620828D0 (en
Inventor
Armstrong Thompson Peter
Edris Badreddin
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Opi Vi - Ip Holdco LLC
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Opi Vi - Ip Holdco LLC
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Priority claimed from US15/173,075 external-priority patent/US20170158772A1/en
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Publication of GB201620828D0 publication Critical patent/GB201620828D0/en
Priority to US15/624,441 priority Critical patent/US20170298139A1/en
Publication of GB2552041A publication Critical patent/GB2552041A/en
Priority to US17/066,776 priority patent/US20210139604A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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  • Immunology (AREA)
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Abstract

A conjugate comprising an immune-stimulatory compound (e.g. an agonist of a TLR, STING or RIG-I) and an antibody construct comprising an antigen binding domain and an Fc domain (i.e. an antibody, wherein the antibody construct may bind CD40) and a linker attaching the antibody construct to the immune stimulatory compound. Also disclosed is a compound of formula (I) and a compound of formula (II): wherein for a compound of formula (I), X1 is OR2 or SR2; X2 is OR3 or SR3; R1, R2, R3 and R4 are hydrogen or a substituent; B1 and B2 are optionally substituted nitrogenous bases; Y is OR4, NR4R4 or halogen; wherein for a compound of formula (II), X1 is OR2 or SR2; X2 is OR3 or SR3; R2, R3 and R4 are hydrogen or a substituent; B1 and B2 are optionally substituted nitrogenous bases; Y is OR4, SR4, NR4R4 or halogen.

Description

(56) Documents Cited:
WO 2017/024296 A1 US 20120231023 A1
A61K 47/68 (2017.01) C07D 401/12 (2006.01) C07D 417/14 (2006.01) C07D 487/04 (2006.01) C07H 21/02 (2006.01) (71) Applicant(s):
OPI VI - IP HoldCo LLC
601 Lexington Ave, 54th Floor, New York 10022, United States of America (72) Inventor(s):
WO 2014/085580 A1 US 20100291109 A1
BMC Cancer, Vol.14, 2014, Khong, A. et al., The efficacy of tumor debulking..., p.969 Toxicol. Letts. Vol.194, 2010, You Qiang, et al., Generation of T cell responses..., pp.79-85 Vaccine, Vol.28, 2010, McWilliams, J. A. et al., Multiple innate signalling pathways..., pp.1468-1476
Peter Armstrong Thompson Badreddin Edris (58) Field of Search:
Other: WPI, EPODOC, BIOSIS, MEDLINE (74) Agent and/or Address for Service:
Forresters IP LLP
Sherborne House, 119-121 Cannon Street, LONDON, EC4N 5AT, United Kingdom (54) Title of the Invention: Compositions of antibody construct-agonist conjugates and methods thereof Abstract Title: Antibody-agonist constructs (57) A conjugate comprising an immune-stimulatory compound (e.g. an agonist of a TLR, STING or RIG-I) and an antibody construct comprising an antigen binding domain and an Fc domain (i.e. an antibody, wherein the antibody construct may bind CD40) and a linker attaching the antibody construct to the immune stimulatory compound. Also disclosed is a compound of formula (I) and a compound of formula (II):
Figure GB2552041A_D0001
wherein for a compound of formula (I), X1 is OR2 or SR2; X2 is OR3 or SR3; R1, R2, R3 and R4 are hydrogen or a substituent; B1 and B2 are optionally substituted nitrogenous bases; Y is OR4, NR4R4 or halogen; wherein for a compound of formula (II), X1 is OR2 or SR2; X2 is OR3 or SR3; R2, R3 and R4 are hydrogen or a substituent; B1 and B2 are optionally substituted nitrogenous bases; Y is OR4, SR4, NR4R4 or halogen.
At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.
1/47
FIGURE ΙΑ
SEQ ID NO: 1:
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGG
TTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATC
TGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACA
GCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTG
ATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGA
AGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTT
CGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCT
GTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT
GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG
GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACA
GAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC
TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC
CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG
TGTTAG
FIGURE IB
SEQ ID NO: 2:
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGG
TTCCAGATGC
FIGURE 1C
SEQ ID NO: 3:
GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGC
CTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATACTG
CATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCT
GGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGC
AACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCGGCGGAGG
GACCAAGGTGGAGATCAA
2/47
FIGURE 2A
SEQ ID NO: 4:
MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWL
AWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY
CQQANIFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQFKSGTASVVCFFNNFYP
REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC
FIGURE 2B
SEQ ID NO: 5:
MRLPAQLLGLLLLWFPGSRC
FIGURE 2C
SEQ ID NO: 6:
DIQMTQSPSSVSASVGDRVTETCRASQGIYSWLAWYQQKPGKAPNLLIYTAS
TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK
3/47
FIGURE 3 A
SEQ ID NO: 7:
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGC
CCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGG
CTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGA
TGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT
CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACAT
GGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGA
GAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTC
GAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCT
CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGG
TCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCA
CGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCG
TTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGG
AGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA
GAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
4/47
Figure 3B
SEQ ID NO: 8:
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGC
CCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGG
CTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGA
TGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT
CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACAT
GGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGA
GAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC
CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC
AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC
ACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
5/47
FIGURE 3C
SEQ ID NO: 9:
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGC
CCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGG
CTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGA
TGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT
CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACAT
GGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGA
GAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGTGGGGGGAC
CGTCAGTCTTCCTCCTGCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCCTGAGGAGCAGTACAACAGCACGCTGCGTGTGGTC
AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
AGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC
CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG
TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG
CAGCCGGAGAACAACTACAAGACCACGCCTCTGGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
6/47
FIGURE 3D
SEQ ID NO: 10:
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGC
CCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGG
CTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGA
TGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT
CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACAT
GGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGA
GAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC
CGGATGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA
GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCGAGGAGAAAACCATC
TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC
CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT
CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC
AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
7/47
FIGURE 3E
SEQ ID NO: 11:
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGC
CCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCT
GGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGG
CTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGA
TGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT
CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACAT
GGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGA
GAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAC
CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACGCCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGCCGCTACCATCTCC
AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA
AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC
CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC
ACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
8/47
FIGURE 3F
SEQ ID NO: 12:
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAG
CCCACTCC
FIGURE 3G
SEQ ID NO: 13:
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT
CAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATA
TGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATG
GATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGC
AGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCT
GAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATC
AGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGG
CCAGGGAACCCTGGTCACCGTCTCCTCAG
9/47
FIGURE 4A
SEQ ID NO: 14:
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG
YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAY
MELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTK
GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE
KTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
TQKSLSLSPGK
10/47
FIGURE 4B
SEQIDNO: 15:
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG
YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAY
MELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTK
GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK
11/47
FIGURE 4C
SEQ ID NO: 16:
MDWTWRIFFFVAAATGAHSQVQFVQSGAEVKKPGASVKVSCKASGYTFT
GYYMHWVRQAPGQGFEWMGWINPDSGGTNYAQKFQGRVTMTRDTSIST
AYMEFNRFRSDDTAVYYCARDQPFGYCTNGVCSYFDYWGQGTFVTVSSA
STKGPSVFPEAPCSRSTSESTAAFGCFVKDYFPEPVTVSWNSGAFTSGVHTF
PAVFQSSGFYSFSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCD
KTHTCPPCPAPEFVGGPSVFFFPPKPKDTEMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVFTVFHQDWFNGKEYKC
KVSNKAFPAPIEKTISKAKGQPREPQVYTEPPSREEMTKNQVSFTCFVKGFY
PSDIAVEWESNGQPENNYKTTPLVFDSDGSFFFYSKFTVDKSRWQQGNVFS
CSVMHEAFHNHYTQKSFSFSPGK
12/47
FIGURE 4D
SEQ ID NO: 17:
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG
YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAY
MELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTK
GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCDKTHTC
PPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK
13/47
FIGURE 4E
SEQIDNO: 18:
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG
YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAY
MELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTK
GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK
14/47
FIGURE 4F
SEQ ID NO: 19:
MDWTWR1LFLVAAATGAHS
FIGURE 4G
SEQ ID NO: 20:
QVQFVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGFEWMG
WINPDSGGTNYAQKFQGRVTMTRDTSISTAYMEFNRFRSDDTAVYYCARDQ
PFGYCTNGVCSYFDYWGQGTFVTVSS
15/47
CLUSTAL 0(1.2.1) multiple sequence alignment
FIGURE 5A
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL SBT-040-G1AAA SB01FJC.wt SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
SBT-040-G1VLPLL
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1DE
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCAG
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCAG
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCAG
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCAG
GTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC
GTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC
GTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC
GTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC
TGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCT
TGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCT
TGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCT
TGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCT
GGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCA
GGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCA
GGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCA
GGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCA
CAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATG
CAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATG
CAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATG
CAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATG
GAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCC
GAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCC
GAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCC
GAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATCAGCCC
CTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTG
CTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTG
CTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTG
CTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTG
GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCC
GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCC
GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCC
GTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCC
AGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
AGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
AGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
AGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
CCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCT
CCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCT
CCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCT
CCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCT
16/47
FIGURE 5B
SBT-040-G1VLPLL GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAAC
SBT-040-G1AAA GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAAC
SBT-040-G1WT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAAC
SBT-040-G1DE GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAAC
SBT-040-G1VLPLL TTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGAC
SBT-040-G1AAA TTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGAC
SBT-040-G1WT TTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGAC
SBT-040-G1DE TTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGAC WWWWWW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
SBT-040-G1VLPLL AAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
SBT-040-G1AAA AAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
SBT-040-G1WT AAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
SBT-040-G1DE AAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
SBT-040-G1VLPLL GAACTCGTGGGGGGACCGTCAGTCTTCCTCCTGCCCCCAAAACCCAAGGACACCCTCATG
SBT-040-G1AAA GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
SBT-040-G1WT GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
SBT-040-G1DE GAACTCCTGGGGGGACCGGArGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG ·+·+·+·+·+·+ ·+·+·+·+·+·+·+·+·+·+·+ ·+·++·+·+·+·+·+·+ ·+ ·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+··+·+·+·+·+·+
SBT-040-G1VLPLL ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
SBT-040-G1AAA ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
SBT-040-G1WT ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
SBT-040-G1DE ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG WWWWWW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
SBT-040-G1VLPLL GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCCT
SBT-040-G1AAA GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
SBT-040-G1WT GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
SBT-040-G1DE GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG **************1**4****************************************
SBT-040-G1VLPLL GAGGAGCAGTACAACAGCACGCTGCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
SBT-040-G1AAA GAGGAGCAGTACAACGCCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
SBT-040-G1WT GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
SBT-040-G1DE GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC ·++·+·+·+·+·+·+·+·+·+·+·+·+·+ ·+·+·+·+ + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + ++ + +
SBT-040-G1VLPLL TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
SBT-040-G1AAA TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
SBT-040-G1WT TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATC
SBT-040-G1DE TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCGAG I a a + + + | +-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-+-A+-+-A
SBT-040-G1VLPLL GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC
SBT-040-G1AAA GCCGCTACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC
SBT-040-G1WT GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC
SBT-040-G1DE GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC +r WW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
SBT-040-G1VLPLL CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
SBT-040-G1AAA CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
SBT-040-G1WT CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
SBT-040-G1DE CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC WWWW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
17/47
FIGURE 5C
SBT-040-G1VLPLL TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
SBT-040-G1AAA TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
SBT-040-G1WT TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
SBT-040-G1DE TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
SBT-040-G1VLPLL ACCACGCCTCTGGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG
SBT-040-G1AAA ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG
SBT-040-G1WT ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG
SBT-040-G1DE ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTG ·+··Α··Α··Α··Α··Α··Α··Α··Α··Α· ·Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Λ··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α·
SBT-040-G1VLPLL GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
SBT-040-G1AAA GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
SBT-040-G1WT GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
SBT-040-G1DE GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
SBT-040-G1VLPLL CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
SBT-040-G1AAA CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
SBT-040-G1WT CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
SBT-040-G1DE CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA ·+··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··Α··+··Α··Α·
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CLUSTAL 0(1.2.1) multiple sequence alignment
SBT-040-G1WT
SBT—040—G1VLPLL SBT—040—G1DE
SBT-040-G1AAA
MDWTWRILFLVAAAT GAH S QVQLVQ S GAEVKKP GASVKVS CKAS GYTF T GYYMHWVRQAP MDWTWRILFLVAAAT GAH S QVQLVQ S GAEVKKP GASVKVS CKAS GYTF T GYYMHWVRQAP MDWTWRILFLVAAAT GAH S QVQLVQ S GAEVKKP GASVKVS CKAS GYTF T GYYMHWVRQAP MDWTWRILFLVAAAT GAH S QVQLVQ S GAEVKKP GASVKVS CKAS GYTF T GYYMHWVRQAP
SBT-040-G1WT
SBT—040—G1VLPLL SBT—040—G1DE
SBT-040-G1AAA
GQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQP
GQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQP
GQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQP
GQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQP
120
120
120
120
WWWWW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
SBT-040-G1WT
SBT-040-G1VLPLL
SBT—040—G1DE
SBT-040-G1AAA
LGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
LGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
LGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
LGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
180
180
180
180
WWWWW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
SBT-040-G1WT
SBT-040-G1VLPLL
SBT—040—G1DE
SBT-040-G1AAA
SBT-040-G1WT
SBT-040-G1VLPLL
SBT—040—G1DE
SBT-040-G1AAA
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD 240 PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD 240 PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD 240 PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVD 240
WWWWWWWWWWWWWWWWWWWWW+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r+r
UHj | LH r—-1 ι 1
KTVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPE 300 KTVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMI SRTPEVTCWVDVSHEDPE 300 KTVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPE 300 KTVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSHEDPE 300 ·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+·+ · +7+7+7 +7+7+7 · +7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7
SBT-040-G1WT
SBT—040—G1VLPLL SBT—040—G1DE
SBT-040-G1AAA
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI VKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPS VKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
360
360
360
360 +7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7 +7+7+7+7+7 +7 +7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7
SBT-040-G1WT
SBT—040—G1VLPLL SBT—040—G1DE
SBT-040-G1AAA
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 420 EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 420 EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 420 AATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 420 +7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7+7
SBT-040-G1WT
SBT-040-G1VLPLL
SBT—040—G1DE
SBT-040-G1AAA
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 475 TTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 475 TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 475 TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 475 +7 +7 +7 + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + +
FIGURE 6
19/47
705
Figure GB2552041A_D0002
Figure GB2552041A_D0003
v
720
FIGURE 7
20/47
805
830
Figure GB2552041A_D0004
820
FIGURE 8
21/47
805
830
Figure GB2552041A_D0005
820
FIGURE 8
22/47
905
930
Figure GB2552041A_D0006
920
FIGURE 9
23/47
Figure GB2552041A_D0007
FIGURE 10
24/47
1105
1180
1185
Figure GB2552041A_D0008
1130
Figure GB2552041A_D0009
1140
Figure GB2552041A_D0010
1120
FIGURE 11
25/47
1245
Figure GB2552041A_D0011
FIGURE 12
26/47
1345
Figure GB2552041A_D0012
FIGURE 13
27/47
1445
Figure GB2552041A_D0013
FIGURE 14
28/47
1545
158
Figure GB2552041A_D0014
FIGURE 15
29/47
Figure GB2552041A_D0015
FIGURE 16
30/47
Figure GB2552041A_D0016
FIGURE 17
31/47
Figure GB2552041A_D0017
FIGURE 18
32/47
Figure GB2552041A_D0018
FIGURE 19
33/47
Figure GB2552041A_D0019
FIGURE 20
34/47
Figure GB2552041A_D0020
FIGURE 21
35/47
Figure GB2552041A_D0021
FIGURE 22
36/47
Figure GB2552041A_D0022
FIGURE 23
37/47
Figure GB2552041A_D0023
FIGURE 24
38/47
Figure GB2552041A_D0024
FIGURE 25
39/47
Figure GB2552041A_D0025
FIGURE 26
40/47
Figure GB2552041A_D0026
FIGURE 27
41/46
DAD1 G, Sig™280,4 Ref™36O,100 (SWS-125-127\ SWS-125-127-RXN2-4EQDTT.D)
Figure GB2552041A_D0027
Area Percent Report
Sorted By : Signal
Multiplier : 1.0000
Dilution : 1.0000
Do not use Multiplier & Dilution Factor with ISTDs
Signal 1 : DAD1 G, Sig=280, 4 Ref=360, 100
Peak # RetTime Type Width Area [mALUs] Height [mAU] Area %
[min] [min] ___________1______________________8 „
1 4.079 a MF B 0.1739 387.76465 37.16523 • J —— — 6.9394
2 5.183 MF 0.2783 183.07185 10.96217 3.2762
3 5.446 MF 0.2503 1014.97418 67.58788 18.1639
4 6.521 MF 0.4789 123.16119 4,28637 2,2041
5 7.261 MF 0.4080 1430.82629 58.44968 25.6060
6 8.726 MF 0.5951 1444.74353 40.46293 25.8551
7 9.962 MF 0.5429 720.32159 22.11237 12.8908
8 10.980 FM 0.6757 282.99246 6.98009 5.0644
FIG. 28
42/46
CB
CM
Figure GB2552041A_D0028
8 co 5Γ CO CM co
¢0 ί o> CM CO 03 o
CD 8 B r- o CO o
w k- B o V- m- T— CM
< ! Ά d !< CN d
8 CM co r~-
S
8 00 M T“ co O)
00 O CO CM
C Σ3 i 00 o h- CO
COS < 1 r- co CM ^j· O
0) F 1 03 03 CM Γ-~ CO
X Lww 8 d co O d
B 8 CM M' CO 'T”
J CD 03 CM CM
«? B CO CO co
* ! if) O co co co
00 CO o CO CM
< I CO M O) CO
< CCS d CM d
5 03 CO v~- co
8 If) CO CM
Figure GB2552041A_D0029
<u
Q.
CO =— t E
CD
03 co CM o co
CO r- co Cf) co
CO 00 co O co
T“ T” co CO io
o d d d d
i-i- ίΐ.. i.!.. ίί.
LL.
co T“ •>M 7™· 0
03 CO CO co M
03 CM cf) CM CO
co M CO CO
2&C a
Φ ! V t\! Γ; \r Lf)
CL !
s
43/46
Figure GB2552041A_D0030
3 1 CO CD
¢0 9 3 cn SO
k.. 0s 9 3 CM 'M' M
< 9 3 CM CM
9 3 CO
3 i 3 o SO
φ,. h- o
JZ 5' i — CM
TO < 1 CD CO
'03 E ’ O CO
I CM U
i 3 CM co
ω co *
Ζ3
Μ
,.<Σ
II
0)
Ω.
0) ε
Η
CC ¢0 -Μ,
CL
to CM
o O
o CO
cn cn
00 r-.
i< x—
cn x—
SO
SO r-
cn <D
’'t CM
Τ- CM
Ο ό
LL LL
S
CM CO
SO M
CO CM
CO
4.496 MF 0.2264 233.63522 17.19782 14.6745
4.723 MF 0.3812 358.71500 15.68383 22.5307
5.213 MF 0.2111 111.90996 6.23586 7.0290 FIG. 30
5.629 MF 0,2375 76.67980 5,38071 4,8162
5.876 FM 0.3740 101.49386 4.52232 6.3748 <\! « ’ΐ sn co s
ΙΟ co
HVm
44/47
FIGURE 31A mDC (donor 358)
Figure GB2552041A_D0031
Cone (ug/mL)
FIGURE 31C
Figure GB2552041A_D0032
SBT-050-WT
SBT-040-WT-A.TAC23
SBT-050-WT-ATAC17
FIGURE 31B
FIGURE 31D mDC (donor 363)
Figure GB2552041A_D0033
Cone (ug/mL) mDC (donor 363)
Figure GB2552041A_D0034
45/47
FIGURE 32A
Figure GB2552041A_D0035
** 1: SBT-050-G2
2: SBT-040-WT-ATAC4
3: SBT-040-WT-ATAC3
4: SBT-040-G2-ATAC4 5: SBT-040-G2-ATAC3
6: SBT-040-AAA-ATAC22
7: SBT-040-VLPLL-ATAC22
8: SBT-040-WT-ATAC1
9: SBT-040-G2-ATAC1
ATAC Cone (ug/mL)
10: SBT-040-WT-ATAC12
11: SBT-040-G2-ATAC12
12: SBT-040-WT-ATAC23
13: SBT-040-AAA-ATAC11
14:SBT-040-VLPLL-ATAC11
15:SBT-040-VLPLL-ATAC12
16:SBT-040-AAA-ATAC12
17:SBT-040-VLPLL-ATAC23
18:SBT-040-AAA-ATAC23 β 19:CD40 Ligand
46/47
FIGURE 32B
Figure GB2552041A_D0036
Figure GB2552041A_D0037
cyoo > <O<0^Cfc
ATAC Cone (ug/mL) ** 1: SBT-050-G2
2: SBT-040-WT-ATAC4
3: SBT-040-WT-ATAC3
4: SBT-040-G2-ATAC4
5: SBT-040-G2-ATAC3
6: SBT-040-AAA-ATAC22
7: SBT-040-VLPLL-ATAC22
8: SBT-040-WT-ATAC1
9: SBT-040-G2-ATAC1
10: SBT-040-WT-ATAC12
11: SBT-040-G2-ATAC12
12: SBT-040-WT-ATAC23
13: SBT-040-AAA-ATAC11
14:SBT-040-VLPLL-ATAC11
15:SBT-040-VLPLL-ATAC12
16:SBT-040-AAA-ATAC12
17:SBT-040-VLPLL-ATAC23
18:SBT-040-AAA-ATAC23 ® 19:CD40 Ligand
47/47
FIGURE 33A
Figure GB2552041A_D0038
LoglOConc. (ug/mL)
SBT-040-ATAC17 * SBT-040-ATAC23 SBT-040-VPLL-ATAC23 • SBT-040-AAA-ATAC23 § SBT-050-WT « Isotype Control
FIGURE 33B
CD83
Figure GB2552041A_D0039
1
-3 -2 -1 0
LoglOConc. (ug/mL) * SBT-050-WT SBT-040-ATAC17 SBT-040-ATAC23 SBT-040-VPLL-ATAC23 SBT-040-AAA-ATAC23 * Isotype Control
FIGURE 33C
Figure GB2552041A_D0040
LoglOConc. (ug/mL)
FIGURE 33
SBT-040-ATAC17 ·*· SBT-040-ATAC23
SBT-040-VPLL-ATAC23 + SBT-040-AAA-ATAC23 s SBT-050-WT * Isotype Control
COMPOSITIONS OF ANTIBODY CONSTRUCT-AGONIST CONJUGATES AND METHODS THEREOF
CROSS REFERENCE [0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/371,141 filed on August 4, 2016, U.S. Patent Application 15/173,075 filed on June 3, 2016, and U.S. Provisional Patent Application No. 62/264,260, filed on December 7, 2015, each of which is incorporated herein by reference in its entirety.
BACKGROUND [0002] One of the leading causes of death in the United States is cancer. The conventional methods of cancer treatment, like chemotherapy, surgery, or radiation therapy, tend to be either highly toxic or nonspecific to a cancer, or both, resulting in limited efficacy and harmful side effects. However, the immune system has the potential to be a powerful, specific tool in fighting cancers. In many cases tumors can specifically express genes whose products are required for inducing or maintaining the malignant state. These proteins may serve as antigen markers for the development and establishment of more specific anti-cancer immune response. The boosting of this specific immune response has the potential to be a powerful anti-cancer treatment that can be more effective than conventional methods of cancer treatment and can have fewer side effects.
INCORPORATION BY REFERENCE [0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
SUMMARY [0004] The composition described herein relates to different embodiments of a conjugate. In various embodiments, a conjugate comprises a) an immune-stimulatory compound; b) an antibody construct comprising an antigen binding domain and an Fc domain, wherein said antigen binding domain binds to a first antigen and wherein a Ka for binding of said Fc domain to an Fc receptor in the presence of said immune-stimulatory compound is no greater than about 100 times a Ka for binding
-1of said Fc domain to said Fc receptor in the absence of the immune stimulatory compound; and c) a linker, wherein said linker attaches said antibody construct to said immune-stimulatory compound.
In some aspects, said antigen binding domain binds said first antigen in a presence of said immunestimulatory compound. In some aspects, a Ka for binding of said antigen binding domain to said first antigen in a presence of said immune-stimulatory compound is less than about 100 nM and no greater than about 100 times a Ka for binding of said antigen binding domain to said first antigen in the absence of said immune-stimulatory compound. In some aspects, said Ka for binding of said antigen binding domain to said first antigen in the presence of said immune-stimulatory compound is less than about lOOnM and is no greater than about 10 times the Ka of the binding of the antigen binding domain to said first antigen in the absence of the immune-stimulatory compound; and said Ka for binding of said Fc domain to said Fc receptor in the presence of said immune-stimulatory compound is no greater than about 10 times said Ka for the binding of said Fc domain to said Fc receptor in the absence of said immune stimulatory compound. In some aspects, a molar ratio of immune-stimulatory compound to antibody construct is less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2.
[0005] In some aspects, said conjugate further comprises a targeting binding domain, wherein said targeting domain is attached to said antibody construct. In some aspects, said targeting binding domain binds a second antigen. In some aspects, said targeting binding domain is attached to said antibody construct at a C-terminal end of said Fc domain.
[0006] In some aspects, said antigen binding domain is from an antibody or non-antibody scaffold. [0007] The conjugate of any of claims 1-9, wherein said antigen binding domain is at least 80% homologous to an antigen binding domain from an antibody or non-antibody scaffold. In some aspects, said non-antibody scaffold is a DARPin, affimer, avimer, knottin, monobody, or affinity clamp. In some aspects, said antigen binding domain is at least 80% homologous to an antigen binding domain from a DARPin, affimer, avimer, knottin, monobody, or affinity clamp.
[0008] In some aspects, said antigen binding domain recognizes a single antigen. In some aspects, said antigen binding domain recognizes two or more antigens. In some aspects, said first antigen is a tumor antigen. In some aspects, said first antigen that is at least 80% homologous to CD5, CD 19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, pl85HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7,
-2EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, poly sialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NYBR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, R0R2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC1, MUC15, MSLN, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, R0R1, MAGE-A3, or Fos-related antigen 1. In some aspects, wherein said first antigen is expressed on an immune cell. In some aspects, said first antigen is expressed on an antigen-presenting cell. In some aspects, said first antigen is expressed on a dendritic cell, a macrophage, or a B-cell. In some aspects, wherein said first antigen is CD40. In some aspects, said antigen binding domain is a CD40 agonist.
[0009] In some aspects, said antibody construct is an antibody. In some aspects, said antibody construct is a human antibody or a humanized antibody. In some aspects, said antibody construct comprises a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 4, at least 80%, 90%, or 100% homologous to SEQ ID NO: 26, or at least 80%, 90%, or 100% homologous to SEQ ID NO: 34. In some aspects, said antibody construct comprises a light chain variable domain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 6. In some aspects, said antibody construct comprises: a) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 15; b) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 16; c) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 17; d) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 18; e) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 22; or f) heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 30. In some aspects, said antibody construct comprises a heavy chain variable domain that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 20. In some aspects, said antibody binding domain comprises at least 80%, 90%, or 100% homology to: a) HC CDR1 comprising an amino acid sequence of SEQ ID NO: 23, HC CDR2 comprising an amino acid sequence of SEQ ID NO: 24, a HC CDR3 comprising an amino acid sequence of SEQ ID NO: 25,
LC CDR1 comprising an amino acid sequence of SEQ ID NO: 27, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 28, and LC CDR3 comprising an amino acid sequence of SEQ ID NO:
-329; or b) HC CDR1 comprising an amino acid sequence of SEQ ID NO: 31, HC CDR2 comprising an amino acid sequence of SEQ ID NO: 32, a HC CDR3 comprising an amino acid sequence of SEQ ID NO: 33, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 35, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 36, and LC CDR3 comprising an amino acid sequence of SEQ ID NO: 37.
[0010] In some aspects, said Fc domain is from an antibody. In some aspects, said Fc domain is at least 80% homologous to an Fc domain from an antibody. In some aspects, said Fc domain binding to said Fc receptor in the presence of said immune-stimulatory compound results in Fc-receptormediated signaling. In some aspects, said Fc domain binding to said Fc receptor in the presence of said immune-stimulatory compound results increased antigen presentation on an immune cell. In some aspects, said Fc domain is a human Fc domain. In some aspects, said Fc domain is selected from a group consisting of a human IgGl Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 Fc domain. In some aspects, wherein said Fc domain is an Fc domain variant comprising at least one amino acid residue change as compared to a wild type sequence of said Fc domain. In some aspects, said Fc domain binds said Fc receptor with altered affinity as compared to a wild type Fc domain. In some aspects, wherein said Fc receptor is selected from a group consisting of CD 16a, CD 16b, CD32a, CD32b, and CD64. In some aspects, said Fc receptor is a CD 16a FI58 variant or a CD 16a VI58 variant. In some aspects, said Fc domain binds said Fc receptor with higher affinity than a wild type Fc domain. In some aspects, said Fc receptor is selected from a group consisting of: CD 16a, CD 16b, CD32a, CD32b, or CD64. In some aspects, said Fc receptor is a CD16a F158 variant or a CD16a V158 variant. In some aspects, said Fc domain has at least one amino acid residue change as compared to wildtype, wherein said at least one amino acid residue change is: a) F243L, R292P, Y300L, L235V, and P396L, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Rabat; b) S239D and I332E, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Rabat; or c) S298A, E333 A, and K334A, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Rabat.
[0011] In some aspects, said immune-stimulatory compound is a damage-associated molecular pattern molecule or a pathogen associated molecular pattern molecule In some aspects, said immunestimulatory compound is a toll-like receptor agonist, STING agonist, or RIG-I agonist. In some aspects, said immune-stimulatory compound is a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, or a TLR10 agonist. In some aspects, said immune-stimulatory compound is selected from a group
-4consisting of: S-27609, CL307, UC-IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS1463GS-9620, GSK2245035, TMX-101, TMX-201, TMX-202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B, and CL663.
[0012] In some aspects, said immune-stimulatory compound comprises one or more rings selected from carbocyclic and heterocyclic rings. In some aspects, said linker is covalently attached to said antibody construct. In some aspects, said linker is covalently attached to said immune-stimulatory compound. In some aspects, said linker is not attached to an amino acid residue of said Fc domain selected from a group consisting of: 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269,
270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286, 288, 290, 291, 292, 293, 294, 295,
296, 297, 298, 299, 300, 302, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330,
331, 332, 333, 334, 335 336, 396, or 428, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Kabat. In some aspects, said linker is attached to an amino acid residue of said antibody construct by a THIOMAB linker, or a Sortase A-catalyzed linker. In some aspects, said linker is attached to said antibody construct via a sulfhydryl group, a primary amine, a hinge cysteine, a Cl lysine, an engineered cysteine in a light chain, an engineered light chain glutamine, or an unnatural amino acid engineered into a light chain or heavy chain. In some aspects, said linker does not interfere with said Fc domain binding to said Fc receptor when said linker is attached to said antibody construct at an amino acid residue. In some aspects, said linker does not interfere with Fc-receptor-mediated signaling resulting from said Fc domain binding to said Fc receptor when said linker is attached to said Fc domain at an amino acid residue. In some aspects, said linker is attached to said immune-stimulatory compound via an exocyclic nitrogen or carbon atom of said immune-stimulatory compound. In some aspects, said immune-stimulatory compound is covalently attached to said linker by a bond to an exocyclic carbon or nitrogen atom on said immune-stimulatory compound.
[0013] In some aspects, wherein said linker is a peptide. In some aspects, said linker is a cleavable linker. In some aspects, said linker selected from a group consisting of: a) a valine-citrulline linker; b) a valine-citrulline linker containing a pentafluorophenyl group; c) a valine-citrulline linker containing a succinimide group; d) a valine-citrulline linker containing a para aminobenzoic acid group; e) a valine-citrulline linker containing a para aminobenzoic acid group and a pentafluorophenyl group; and f) a valine-citrulline linker containing a para aminobenzoic acid group and a succinimide group.
-5[0014] In some aspects, said linker is a non-cleavable linker. In some aspects, said linker selected from a group consisting of: a) a maleimidocaproyl linker; b) a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules; c) a maleimide-PEG4 linker; d) a maleimidocaproyl linker containing a succinimide group; e) a maleimidocaproyl linker containing a pentafluorophenyl group; f) a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules; and g) a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules.
[0015] In some aspects, said conjugate induces the secretion of cytokine by an antigen presenting cell. In some aspects, said cytokine is IFN-γ, IL-8, IL-12, IL-2, or a combination thereof. In some aspects, said conjugate induces antigen presentation on an antigen presenting cell.
[0016] In some aspects, said conjugate is formulated to treat tumors.
[0017] In some aspects, wherein said conjugate is in a pharmaceutical formulation.
[0018] In some embodiments, a pharmaceutical composition comprises said conjugate of any of the proceeding embodiments and a pharmaceutically acceptable carrier.
[0019] In some embodiments, a method of producing the conjugate of any of the preceding embodiments, comprises: a) selecting an antibody construct; b) selecting an immune-stimulatory compound; and c) attaching said antibody construct to said immune-stimulatory compound, wherein said immune-stimulatory compound is attached to said antibody construct via a linker and said antibody construct comprises an antigen binding domain and an Fc domain, wherein said antigen binding domain specifically binds an antigen in the presence of said immune-stimulatory compound and said Fc domain specifically binds an Fc receptor in the presence of said immune-stimulatory compound.
[0020] In some embodiments, a method of producing the conjugate of any of the preceding embodiments, comprises: a) selecting an antibody construct; b) selecting an immune-stimulatory compound; c) selecting a targeting binding domain; d) attaching said targeting binding domain to said antibody construct; and e) attaching said antibody construct to said immune-stimulatory compound, wherein said immune-stimulatory compound is attached to said antibody construct via a linker, wherein said antigen binding domain specifically binds a first antigen in the presence of said immune-stimulatory compound and said targeting binding specifically binds a second antigen in the presence of said immune-stimulatory compound.
[0021] In some embodiments, a method for treating a subject in need thereof, comprises administering a therapeutic dose of said conjugate of any one of the preceding embodiments or said pharmaceutical composition of any of the preceding embodiments. In some aspects, said subject has -6cancer. In some aspects, said composition is administered intravenously, cutaneously, subcutaneously, or injected at a site of affliction.
[0022] In some embodiments, a kit comprises said conjugate of any of the preceding embodiments. [0023] A composition comprising a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 4 and heavy chain sequence that is at least 80%, 90%, or 100% [0024] A composition comprising:
a) a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 4 or at least 80%, 90%, or 100% homologous to SEQ ID NO: 26; and
b) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 16, at least 80%, 90%, or 100% homologous to SEQ ID NO: 17, or at least 80%, 90%, or 100% homologous to SEQ ID NO: 18.
[0025] The composition of claim 75, wherein said composition binds to an Fc receptor with greater affinity than an antibody comprising a heavy chain sequence of SEQ ID NO: 15 or SEQ ID NO: 22.
[0026] .
[0027] In some aspects, the present disclosure provides a compound represented by the structure of Formula (I):
Figure GB2552041A_D0041
or a pharmaceutically acceptable salt thereof, wherein:
X1 is selected from -OR2 and -SR2;
X2 is selected from -OR3 and -SR3;
B and B are independently selected from optionally substituted nitrogenous bases;
Y is selected from —OR4, -NR4R4, and halogen;
R1, R2, R3 and R4 are independently selected at each occurrence from hydrogen, -C(=O)R100, C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -7N02, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R , R , R and R is independently optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -0P(0)(0Rw°)2, -CN, Ci^ alkyl, C2.6 alkenyl, and C2.6 alkynyl; and R100 at each occurrence is independently selected from hydrogen; and Cmo alkyl, C2_io alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -N02, =0, =S, and haloalkyl.
[0028] In some embodiments, the compound of Formula (I) is represented by Formula (IA):
Figure GB2552041A_D0042
or pharmaceutically acceptable salts thereof.
[0029] In an alternative embodiment, the compound of Formula (I) is represented by Formula (IB):
Figure GB2552041A_D0043
(IB) or a pharmaceutically acceptable salt thereof.
2 [0030] In various embodiments, B and B are independently selected from optionally substituted purines, such as optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, and optionally substituted isoguanine. In a -81 2 preferred embodiment, B and B are independently selected from optionally substituted adenine and optionally substituted guanine.
2 [0031] In some embodiments, B and B are independently optionally substituted with one or more
2 substituents, wherein the optional substituents on B and B are independently selected at each occurrence from halogen, =0, =S, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, -P(O)(OR100)2, -OP(O)(OR100)2 and -CN; Cmo alkyl, C2. io alkenyl, C2_io alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle;
and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to
10-membered heterocycle is independently optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci^ alkyl, C2.6 alkenyl,
2 and C2_6 alkynyl. In a preferred embodiment, B and B are independently optionally substituted with one or more substituents, wherein the optional substituents on B and B are independently selected at each occurrence from halogen, =0, =S, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, C(O)R100, -C(O)OR100, -OC(O)R100, -N02, -P(O)(OR100)2, -OP(O)(OR100)2, -CN and Cmo alkyl. In some embodiments, B is an optionally substituted guanine. In some embodiments, B is an optionally substituted guanine.
[0032] In various embodiments, X1 is selected from -OH and -SH. For example, X1 may be -OH. In various embodiments, X is selected from -OH and -SH. For example, X may be -OH.
[0033] In various embodiments, b' is selected from OH, O-Ci-io alkyl, biH(Ci_io alkyl), and bIH2. For example, Y may be -OH.
[0034] In various embodiments, R100 is independently selected at each occurrence from hydrogen and Cmo alkyl optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO2, =0, and =S.
[0035] In various embodiments, the compound of Formula (I) is represented by Formula (IC):
-9HO C H2NrNyN hnY^n
Figure GB2552041A_D0044
, ^Λη > N N Nh2
HO
Figure GB2552041A_D0045
O NH2 (1C) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (IC) is represented by Formula (ID):
O
OH H2NyNyN o
HO O
Figure GB2552041A_D0046
Figure GB2552041A_D0047
ZO NH2 /o
HO
N-< NH
I I' — y n nh2 (ID) or a pharmaceutically acceptable salt thereof.
[0036] In various embodiments, the compound is a pharmaceutically acceptable salt. The compound or salt may agonize a stimulator of interferon genes (STING).
[0037] In some aspects, the present disclosure provides an antibody drug conjugate, comprising a compound or salt previously described, an antibody, and a linker group, wherein the compound or salt is linked to the antibody through the linker group. The linker group may be selected from a cleavable or non-cleavable linker.
[0038] In some aspects, the present disclosure provides a compound represented by the structure of Formula (II):
Figure GB2552041A_D0048
or a pharmaceutically acceptable salt thereof, wherein:
(II)
-10X1 is selected from -OR2 and -SR2;
X2 is selected from -OR3 and -SR3;
B and B are independently selected from optionally substituted nitrogenous bases, wherein each optional substituent is independently selected from halogen, -OR100, -SR100, N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, R6, and -X3;
Y is selected from —OR4, -SR4,-NR4R4, and halogen;
Z is selected from —OR5, -SR5, and -NR5R5;
R1, R2, R3, R4, and R5 are independently selected from a -X3; hydrogen, -C(=O)R100, C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2_io alkenyl, C2_io alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, OC(O)R100, -N02, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R , R , R , R , and R is optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, N(R100)2, -S(O)R100, -S(0)2R100-C(0)R100, -C(O)OR100, -OC(O)R100, -N02, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(ORw°)2, -CN, Cm alkyl, Cm alkenyl, Cm alkynyl;
R6is independently selected from -C(=O)R100, -C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2-io alkenyl, C2_io alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(ORw°)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R6 is optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100C(O)R100, -C(O)OR100, -OC(O)R100, -N02, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(ORw°)2, -CN, Cm alkyl, Cm alkenyl, Cm alkynyl;
R100 at each occurrence is independently selected from hydrogen; and Cmo alkyl, C2. 10 alkenyl, C2_io alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -N02, =0, =S, and haloalkyl; and
-113 12345121 2
X is a linker moiety, wherein at least one ofR,R,R,R,R,X,X,aB substituent and a B β
substituent is -X .
[0039] In various embodiments, the compound of Formula (II) is represented by a structure of Formula (IIA):
Figure GB2552041A_D0049
(HA) or pharmaceutically acceptable salts thereof.
[0040] In various embodiments, the compound of Formula (II) is represented by a structure of Formula (IIB):
Figure GB2552041A_D0050
[0041] or a pharmaceutically acceptable salt thereof.
2 [0042] In various embodiments, B and B are independently selected from optionally substituted
2 purines. B and B may be each, independently selected from one another, adenine, guanine, and derivatives thereof. B and B may be independently selected from optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, and optionally substituted isoguanine. In a preferred embodiment, B and B are independently selected from optionally substituted adenine and optionally substituted guanine.
3 [0043] In various embodiments, B is substituted by X and optionally one or more additional substituents independently selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, and R6. For example, -12ΗΝ
Ϋ Ν 1
Β may be represented by: «»ν , and wherein B is optionally further substituted by one or more substituents.
3 [0044] In various embodiments, B is substituted by X and optionally one or more additional substituents independently selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, and R6. For example,
HN
X'
I
B may be represented by: »/wv more substituents.
, and wherein B is optionally further substituted by one or
3 3 1 [0045] In various embodiments, X is selected from -Ο- X and-S-X . In some embodiments, X is selected from -OH and -SH.
3 3 2 [0046] In various embodiments, X is selected from -Ο- X and-S-X . In some embodiments, X is selected from -OH and -SH.
3 3 3 [0047] In various embodiments, Y is selected from -NR X , -S-X , and -Ο- X . In some embodiments, Y is selected from -OH, -SH, -O-Cmo alkyl, -NH(Cmo alkyl), and -NH2
3 3 3 [0048] In various embodiments, Z is selected from -NR X , -S-X , and -Ο- X . In some embodiments, Z is selected from -OH, -SH, -O-Cmo alkyl, -NH(Cmo alkyl), and -NH2 β
[0049] In various embodiments, -X is a represented by the formula:
β [0050] In some embodiments, -X is represented by the formula: wherein RX comprises a reactive moiety, such a maleimide.
[0051]
Figure GB2552041A_D0051
Λ
N peptide H
Figure GB2552041A_D0052
O χ
N peptide—RX
H
-13β [0052] In some embodiments, -X is represented by the formula:
Λ
N peptide-RX*-Antibody *
H , wherein RX is a reactive moiety that has reacted with a moiety on an antibody to form an antibody drug conjugate.
β [0053] In some embodiments, -X is represented by the formula:
O O '^XX^'RX
T4 H , wherein RX is a reactive moiety, such as a maleimide.
β [0054] In some embodiments, -X is represented by the formula:
O O < yo / Ο N RX*-Antibody
Ί-4 H , wherein RX is a reactive moiety that has reacted with a moiety on an antibody to form an antibody drug conjugate.
[0055] In some embodiments, the compound is represented by the formula:
/
Ν'Α'··<μ ρ·°Ά p/O HN' xiP nAnAnh2 ho φΑ
Figure GB2552041A_D0053
h2n^n,
HN.
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
h2nn
HN
Figure GB2552041A_D0054
</Nl P
HO^O Yn Nhfe
Figure GB2552041A_D0055
o-p;
oh o o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
-14, or a pharmaceutically acceptable salt thereof.
[0056] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0056
θ , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0057
CHs , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0058
H CH3 O or a pharmaceutically acceptable salt thereof.
[0057] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0059
, or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
, or a pharmaceutically acceptable salt
Figure GB2552041A_D0060
thereof. The compound may be represented by the formula:
Figure GB2552041A_D0061
0 , or a pharmaceutically acceptable salt thereof.
[0058] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0062
θ , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0063
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0064
Ο , or a pharmaceutically acceptable salt thereof.
[0059] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0065
, or a pharmaceutically acceptable salt thereof. The compound is represented by the formula:
Figure GB2552041A_D0066
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0067
o , or a pharmaceutically acceptable salt thereof.
-17[0060] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0068
θ , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0069
, or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0070
Figure GB2552041A_D0071
o , or a pharmaceutically acceptable salt thereof.
[0061] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0072
° , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
-18, or a pharmaceutically
Figure GB2552041A_D0073
actable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0074
Figure GB2552041A_D0075
o , or a pharmaceutically acceptable salt thereof.
[0062] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0076
thereof. The compound may be represented by the formula:
, or a pharmaceutically acceptable salt
Figure GB2552041A_D0077
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
-19, or a pharmaceutically acceptable salt
Figure GB2552041A_D0078
o thereof.
[0063] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0079
thereof. The compound may be represented by the formula:
, or a pharmaceutically acceptable salt
Figure GB2552041A_D0080
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0081
o , or a pharmaceutically acceptable salt thereof.
-20BRIEF DESCRIPTION OF THE DRAWINGS [0064] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
[0065] FIGURE 1A illustrates a DNA sequence (SEQ ID NO: 1) of a light chain of a human CD40 monoclonal antibody SBT-040. Furthermore, SEQ ID NO: 1 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 2) as shown in FIGURE IB and a variable domain sequence (SEQ ID NO: 3) as shown in FIGURE 1C.
[0066] FIGURE IB illustrates a DNA sequence of a signal sequence (SEQ ID NO: 2) of a light chain of a human CD40 monoclonal antibody SBT-040.
[0067] FIGURE 1C illustrates a DNA sequence of a variable domain (SEQ ID NO: 3) in a light chain of a human CD40 monoclonal antibody SBT-040.
[0068] FIGURE 2A illustrates an amino acid sequence (SEQ ID NO: 4) of a light chain of a human CD40 monoclonal antibody SBT-040. Furthermore, SEQ ID NO: 4 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO: 5) as shown in FIGURE 2B and a variable domain sequence (SEQ ID NO: 6) as shown in FIGURE 2C.
[0069] FIGURE 2B illustrates an amino acid sequence of a signal sequence (SEQ ID NO: 5) of a light chain of a human CD40 monoclonal antibody SBT-040.
[0070] FIGURE 2C illustrates an amino acid sequence of a variable domain (SEQ ID NO: 6) in a light chain of a human CD40 monoclonal antibody SBT-040.
[0071] FIGURE 3A illustrates a DNA sequence (SEQ ID NO: 7) of a wildtype IgG2 isotype heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G2. Furthermore, SEQ ID NO: 7 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0072] FIGURE 3B illustrates a DNA sequence (SEQ ID NO: 8) of a wild type IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G1WT. Furthermore, SEQ ID NO: 8 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0073] FIGURE 3C illustrates a DNA sequence (SEQ ID NO: 9) of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing DNA nucleotide modifications
-21corresponding to L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications of a wild type IgGl Fc domain, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G1VLPLL. The modified DNA nucleotides corresponding to the L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 9 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0074] FIGURE 3D illustrates a DNA sequence (SEQ ID NO: 10) of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing DNA nucleotide modifications corresponding to S239D and I332E amino acid residue modifications of a wild type IgGl Fc domain, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040G1DE. The modified DNA nucleotides corresponding to the S239D and I332E amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 10 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0075] FIGURE 3E illustrates a DNA sequence (SEQ ID NO: 11) of an IgGl isotype heavy chain of human CD40 monoclonal antibody SBT-040 containing DNA nucleotide modifications corresponding to S298A, E333A, and K334A amino acid residue modifications of a wild type IgGl Fc domain, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040G1 AAA. The modified DNA nucleotides corresponding to the S298A, E333A, and K334A amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 11 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0076] FIGURE 3F illustrates a DNA sequence of a signal sequence (SEQ ID NO: 12) of a heavy chain of a human CD40 monoclonal antibody SBT-040.
[0077] FIGURE 3G illustrates a DNA sequence of a variable domain (SEQ ID NO: 13) in a heavy chain of a human CD40 monoclonal antibody SBT-040.
[0078] FIGURE 4A illustrates an amino acid sequence (SEQ ID NO: 14) of a wildtype IgG2 isotype heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G2. Furthermore, SEQ ID NO: 14 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO: 19) as shown in FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE 4G.
[0079] FIGURE 4B illustrates an amino acid sequence (SEQ ID NO: 15) of a wild type IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this heavy chain of
-22the SBT-040 antibody can also be referred to as SBT-040-G1WT. Furthermore, SEQ ID NO: 15 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO: 19) as shown in FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE 4G.
[0080] FIGURE 4C illustrates an amino acid sequence (SEQ ID NO: 16) of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications of a wild type IgGl Fc domain, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G1VLPLL. The amino acid residues corresponding to the L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 16 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO: 19) as shown in FIGURE 4F and a variable domain sequence (SEQ ID NO: 15) as shown in FIGURE 4G.
[0081] FIGURE 4D illustrates an amino acid sequence (SEQ ID NO: 17) of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing S239D and 1332 amino acid residue modifications of a wild type IgGl Fc domain, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G1DE. The amino acid residues corresponding to the S239D and I332E amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 17 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO: 19) as shown in FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE 4G.
[0082] FIGURE 4E illustrates an amino acid sequence (SEQ ID NO: 18) of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing S298A, E333A, and K334A amino acid residue modifications of a wild type IgGl Fc domain, wherein this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-G1 AAA. The amino acid residues corresponding to the S298A, E333A, and K334A amino acid modifications are in bold. Furthermore, SEQ ID NO: 11 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO: 19) as shown in FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE 4G. [0083] FIGURE 4F illustrates an amino acid sequence of a signal sequence (SEQ ID NO: 19) of a heavy chain of a human CD40 monoclonal antibody SBT-040.
[0084] FIGURE 4G illustrates an amino acid sequence of a variable domain (SEQ ID NO: 20) in a heavy chain of a human CD40 monoclonal antibody SBT-040.
[0085] FIGURES 5A, 5B, & 5C illustrate a CLUSTAL 0(1.2.1) multiple DNA sequence alignment of the DNA sequences of SBT-040-G1 VLPLL (SEQ ID NO: 9), SBT-040-G1AAA (SEQ ID NO:
11), SBT-040-G1WT (SEQ ID NO: 8), and SBT-040-G1DE (SEQ ID NO: 10). The SBT-040G1VLPLL sequence is a DNA sequence of an IgGl isotype heavy chain of a human CD40
-23monoclonal antibody SBT-040 containing DNA nucleotide modifications corresponding to L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications of a wild type IgGl Fc domain. The modified DNA nucleotides corresponding to the F235V, F243F, R292P, Y300F, and P396F amino acid residue modifications are in bold. The SBT-040-G1 AAA sequence is a DNA sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing DNA nucleotide modifications corresponding to S298A, E333A, and K334A amino acid residue modifications of a wild type IgGl Fc domain. The modified DNA nucleotides corresponding to the S298A, E333A, and K334A amino acid residue modifications are boxed. The SBT-040-G1WT sequence is a DNA sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040. The SBT-040-G1 AAA sequence is a DNA sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing DNA nucleotide modifications corresponding to S239D and I332E amino acid residue modifications of a wild type IgGl Fc domain. The modified DNA nucleotides corresponding to the S239D and I332E amino acid residue modifications are in bold italics. FIGURE 5A shows the start of the sequence alignment. FIGURE 5B shows the middle of the sequence alignment as a continuation of FIGURE 5A. FIGURE 5C shows the end of the sequence alignment as continuation of FIGURE 5C.
[0086] FIGURE 6 illustrates a CFUSTAF 0(1.2.1) multiple amino acid sequence alignment of the amino acid sequences of SBT-040-G1VFPFF (SEQ ID NO: 16), SBT-040-G1AAA (SEQ ID NO: 18), SBT-040-G1WT (SEQ ID NO: 15), and SBT-040-G1DE (SEQ ID NO: 17). The SBT-040G1VLPLL sequence is an amino acid sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications of a wild type IgGl Fc domain. The L235V, F243L, R292P, Y300F, and P396F amino acid residue modifications are in bold. The SBT-040-G1 AAA sequence is an amino acid sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing S298A, E333A, and K334A amino acid residue modifications of a wild type IgGl Fc domain. The S298A, E333A, and K334A amino acid residue modifications are italics. The SBT040-G1WT sequence is an amino acid sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040. The SBT-040-G1AAA sequence is an amino acid sequence of an IgGl isotype heavy chain of a human CD40 monoclonal antibody SBT-040 containing S239D and I332E amino acid residue modifications bold italics. Additionally, the hinge region of each amino acid sequence is differentiated from other regions of the amino acid sequence by brackets. The left bracket indicates the upper portion of the hinge region (UH). The four residues between the brackets
-24are the middle portion of the hinge region. The right bracket indicates the lower portion of the hinge region (LH).
[0087] FIGURE 7 illustrates a schematic of an antibody. An antibody contains two heavy chains as shown in gray and two light chains as shown in light gray. A portion of the heavy chains contain Fc domains (705 and 720). An antibody contains two antigen binding sites (710 and 715).
[0088] FIGURE 8 illustrates a schematic of an exemplary conjugate. An antibody construct is an antibody, which contains two heavy chains as shown in gray and two light chains as shown in light gray. The antibody comprises two antigen binding sites (810 and 815), and a portion of the heavy chains contain Fc domains (805 and 820). The immune-stimulatory compounds (830 and 840) are conjugated to the antibody by linkers (860 and 870).
[0089] FIGURE 9 illustrates a schematic of an exemplary conjugate. An antibody construct is an antibody, which contains two heavy chains as shown in gray and two light chains as shown in light gray. The antibody comprises two antigen binding sites (910 and 915), and a portion of the heavy chains contain Fc domains (905 and 920). The immune-stimulatory compounds (930 and 940) are conjugated to the antibody by linkers (960 and 970). Targeting binding domains are conjugated to the antibody (980 and 985).
[0090] FIGURE 10 illustrates a schematic of an exemplary conjugate. An antibody construct contains the Fc region of an antibody with the heavy chains shown in gray, and two scaffolds as shown in light gray. The antibody construct comprises two antigen binding sites (1010 and 1015) in the scaffolds, and a portion of the heavy chains contain Fc domains (1005 and 1020). The immunestimulatory compounds (1030 and 1040) are conjugated to the antibody construct by linkers (1060 and 1070).
[0091] FIGURE 11 illustrates a schematic of an exemplary conjugate. An antibody construct contains the Fc region of an antibody with the heavy chains shown in gray, and two scaffolds as shown in light gray. The antibody construct comprises two antigen binding sites (1110 and 1115) in the scaffolds, and a portion of the heavy chains contain Fc domains (1105 and 1120). The immunestimulatory compounds (1130 and 1140) are conjugated to the antibody construct by linkers (1160 and 1170). Targeting binding domains are conjugated to the antibody construct (1180 and 1185). [0092] FIGURE 12 illustrates a schematic of an exemplary conjugate. An antibody construct contains the F(ab')2 region of an antibody with heavy chains shown in gray and light chains shown in light gray, and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1210 and 1215), and a portion of two scaffolds contain Fc domains (1220 and 1245).
-25The immune-stimulatory compounds (1230 and 1240) are conjugated to the antibody construct by linkers (1260 and 1270).
[0093] FIGURE 13 illustrates a schematic of an exemplary conjugate. An antibody construct contains the F(ab')2 region of an antibody with heavy chains shown in gray and light chains shown in light gray, and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1310 and 1315), and a portion of two scaffolds contain Fc domains (1320 and 1345). The immune-stimulatory compounds (1330 and 1340) are conjugated to the antibody construct by linkers (1360 and 1370). Targeting binding domains are conjugated to the antibody construct (1380 and 1385).
[0094] FIGURE 14 illustrates a schematic of an exemplary conjugate. An antibody construct contains two scaffolds as shown in light gray and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1410 and 1415), and a portion of the two dark gray scaffolds contain Fc domains (1420 and 1445). The immune-stimulatory compounds (1430 and 1440) are conjugated to the antibody construct by linkers (1460 and 1470).
[0095] FIGURE 15 illustrates a schematic of an exemplary conjugate. An antibody construct contains two scaffolds as shown in light gray and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1510 and 1515), and a portion of the two dark gray scaffolds contain Fc domains (1520 and 1545). The immune-stimulatory compounds (1530 and 1540) are conjugated to the antibody construct by linkers (1560 and 1570). Targeting binding domains are conjugated to the antibody construct (1580 and 1585).
[0096] FIGURE 16 is the two-dimensional structure of the heavy chain of Dacetuzumab.
[0097] FIGURE 17 is the two-dimensional structure of the light chain of Dacetuzumab.
[0098] FIGURE 18 is the two-dimensional structure of the heavy chain of Bleselumab.
[0099] FIGURE 19 is the two-dimensional structure of the light chain of Bleselumab.
[0100] FIGURE 20 is the two-dimensional structure of the heavy chain of Lucatumumab.
[0101] FIGURE 21 is the two-dimensional structure of the light chain of Lucatumumab [0102] FIGURE 22 is the two-dimensional structure of the heavy chain of ADC-1013.
[0103] FIGURE 23 is the two-dimensional structure of the light chain of ADC-1013.
[0104] FIGURE 24 is the two-dimensional structure of the heavy chain of humanized rabbit antibody APX005.
[0105] FIGURE 25 is the two-dimensional structure of the light chain of humanized rabbit antibody APX005.
[0106] FIGURE 26 is the two-dimensional structure of the heavy chain of Chi Lob 7/4.
-26[0107] FIGURE 27 is the two-dimensional structure of the light chain of Chi Lob 7/4.
[0108] FIGURE 28 shows HPLC analysis of SBT-040-G1WT conjugated to a Cys-targeted drug linker tool compound.
[0109] FIGURE 29 shows HPLC analysis of SBT-040-G1WT conjugated to ATAC2.
[0110] FIGURE 30 shows HPLC analysis of SBT-040-G2WT conjugated to ATAC2.
[0111] FIGURE 31A shows the concentration of IL-12p70 produced by dendritic cells (DCs) from donor 358 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with SBT-050-WT.
[0112] FIGURE 31B shows the concentration of IL-12p70 produced by DCs from donor 363 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with SBT-050-WT [0113] FIGURE 31C shows the concentration of TNLa produced by DCs from donor 358 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with SBT-050-WT [0114] FIGURE 31D shows the concentration of TNLa produced by DCs from donor 363 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with SBT-050-WT [0115] FIGURE 32A shows the concentration of IL-12p70 produced by DCs after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT040-AAA-ATAC22, SBT-040-VLPLL-ATAC22, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30, SBT-040-G1AAAATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT 040-VLPLL-ATAC23, and SBT-040-AAA-ATAC23 as compared with SBT-050-G2 or CD40 ligand.
[0116] FIGURE 32B shows the concentration of IL-6 produced by DCs from donor 2 after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040G2-ATAC3, SBT-040-AAA-ATAC22, SBT-040-VLPLL-ATAC22, SBT-040-WT-ATAC1, SBT040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30, SBT040-AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAAATAC12, SBT-040-VLPLL-ATAC23, and SBT-040-AAA-ATAC30 compared with SBT-050-G2 or CD40 ligand. Results are shown for the immune stimulatory cytokines IL-12p70 and IL-6.
[0117] FIGURE 33A shows a dose dependent increase in CD86 expression on dendritic cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLL-ATAC22, SBT040-AAA-ATAC23 as compared to treatment a control SBT-050-WT or staining with an isotype control.
-27[0118] FIGURE 33B shows a dose dependent increase in CD83 expression on dendritic cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040- VLPLL-ATAC23, SBT040-AAA-ATAC23 as compared to treatment a control SBT-050-WT or staining with an isotype control.
[0119] FIGURE 33C shows a dose dependent increase in MHC class II expression on dendritic cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLLATAC23, SBT-040-AAA-ATAC23 as compared to treatment a control SBT-050-WT or staining with an isotype control.
DETAILED DESCRIPTION [0120] Additional aspects and advantages of the present disclosure will become apparent to those skilled in this art from the following detailed description, wherein illustrative aspects of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different aspects, and its several details are capable of modifications in various respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
[0121] Cancer is one of the leading causes of death in the United States. Conventional methods of cancer treatment like chemotherapy, surgery or radiation therapy, can be limited in their efficacy since they are often nonspecific to the cancer. In many cases tumors, however, can specifically express genes whose products are required for inducing or maintaining the malignant state. These proteins may serve as antigen markers for the development and establishment of efficient anti-cancer treatments.
[0122] As used herein, “homologous” or “homology” can refer to the similarity between a DNA, RNA, nucleotide, amino acid, or protein sequence to another DNA, RNA, nucleotide, amino acid, or protein sequence. Homology can be expressed in terms of a percentage of sequence identity of a first sequence to a second sequence. Percent (%) sequence identity with respect to a reference DNA sequence can be the percentage of DNA nucleotides in a candidate sequence that are identical with the DNA nucleotides in the reference DNA sequence after aligning the sequences. Percent (%) sequence identity with respect to a reference amino acid sequence can be the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. As used herein, the term “antibody” can refer to an immunoglobulin molecule that specifically binds to,
-28or is immunologically reactive toward, a specific antigen. Antibody can include, for example, polyclonal, monoclonal, genetically engineered, and antigen binding fragments thereof. An antibody can be, for example, murine, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody. The antigen binding fragment can include, for example, Fab', F(ab')2, Fab, Fv, rlgG, and scFv.
[0123] As used herein, “recognize” can refer to the association or binding between an antigen binding domain and an antigen.
[0124] As used herein, a “tumor antigen” can be an antigenic substance associated with a tumor or cancer cell, and can trigger an immune response in a host.
[0125] As used herein, an “antibody construct” can refer to a construct that contains an antigen binding domain and an Fc domain.
[0126] As used herein, an “antigen binding domain” can refer to an antigen binding domain from an antibody or from a non-antibody that can bind to the antigen.
[0127] As used herein, a “Fc domain” can be an Fc domain from an antibody or from a non-antibody that can bind to an Fc receptor.
[0128] As used herein, a “target binding domain” can refer to a construct that contains an antigen binding domain from an antibody or from a non-antibody that can bind to the antigen.
[0129] The term “salt” or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically
-29acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
[0130] The term “Cx.y” when used in conjunction with a chemical moiety, such as alkyl, alkenyl, or alkynyl is meant to include groups that contain from x to y carbons in the chain. For example, the term “Cx.yalkyl” refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.
[0131] The terms “Cx.yalkenyl” and “Cx.yalkynyl” refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
[0132] The term “carbocycle” as used herein refers to a saturated, unsaturated or aromatic ring in which each atom of the ring is carbon. Carbocycle includes 3- to 10-membered monocyclic rings, 6to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, and naphthyl.
[0133] The term “heterocycle” as used herein refers to a saturated, unsaturated or aromatic ring comprising one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6to 12-membered bridged rings. Each ring of a bicyclic heterocycle may be selected from saturated, unsaturated, and aromatic rings wherein at least one of the rings includes a heteroatom. In an exemplary embodiment, an aromatic ring, e.g., pyridyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, morpholine, piperidine or cyclohexene. The term “heteroaryl” includes aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term “heteroaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be aromatic or non-aromatic carbocyclic, or heterocyclic. Heteroaryl groups include, for example,
-30pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
[0134] Th e term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., NH, of the structure. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In certain embodiments, substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acycli c and cycli c, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
[0135] In some embodiments, substituents may include any substituents described herein, for example: halogen, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazino (=NNH2), -Rb-0Ra, -Rb-OC(O)-Ra, -Rb-OC(O)-ORa, -Rb-OC(O)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(O)Ra, -Rb-C( O)ORa, -Rb-C(O)N(Ra)2, -Rb-0-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-N(Ra)C(0)Ra, -Rb-N(Ra)S( O)tRa (where t is 1 or 2), -Rb-S(O)tRa (where t is 1 or 2), -Rb-S(O)tORa (where t is 1 or 2), and -Rb-S(O)tN(Ra)2 (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl any of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-0H), hydrazine (=NNH2), -Rb-0Ra, -Rb-0C(0)-Ra, -Rb-0C(0)-0Ra, -Rb-0C(0)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(0)Ra, -Rb-C( O)ORa, -Rb-C(0)N(Ra)2, -Rb-0-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-N(Ra)C(0)Ra, -Rb-N(Ra)S( O)tRa (where t is 1 or 2), -Rb-S(O)tRa (where t is 1 or 2), -Rb-S(O)tORa (where t is 1 or 2) and -Rb-S(O)tN(Ra)2 (where t is 1 or 2); wherein each Ra is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl,
-31or heteroarylalkyl, wherein each Ra, valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H), oximo (=N-0H), hydrazine (=NNH2), -Rb-0Ra, -Rb-0C(0)-Ra, -Rb-0C(0)-0Ra, -Rb-0C(0)-N(Ra)2, -Rb-N(Ra)2, -Rb-C(0)Ra, -Rb-C( 0)0Ra, -Rb-C(0)N(Ra)2, -Rb-0-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-N(Ra)C(0)Ra, -Rb-N(Ra)S( 0)tRa (where t is 1 or 2), -Rb-S(O)tRa (where t is 1 or 2), -Rb-S(O)tORa (where t is 1 or 2) and -R -S(O)tN(R )2 (where t is 1 or 2); and wherein each R is independently selected from a direct bond or a straight or branched alkylene, alkenylene, or alkynylene chain, and each Rc is a straight or branched alkylene, alkenylene or alkynylene chain.
[0136] It will be understood by those skilled in the art that substituents can themselves he substituted, if appropriate. Unless specifically stated as “unsubstituted,” references to chemical moieties herein are understood to include substituted variants. For example, reference to a “heteroaryl” group or moiety implicitly includes both substituted and unsubstituted variants.
[0137] Chemical entities having carbon-carbon double bonds or carbon-nitrogen double bonds may exist in Z- or E- form (or cis- or trans- form). Furthermore, some chemical entities may exist in various tautomeric forms. Unless otherwise specified, chemical entities described herein are intended to include all Z-, E- and tautomeric forms as well.
[0138] A tautomer refers to a molecule wherein a proton shift from one atom of a molecule to another atom of the same molecule is possible. The compounds presented herein, in certain embodiments, exist as tautomers. In circumstances where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors,
-32including physical state, temperature, solvent, and pH. Some examples of tautomeric equilibrium
OH — H \Αλ Η H \Λλ H
O __ XA \ nh2 OH \Ah nh2 XA λ - = NH xA λ \ N ' ' H
H N —= N'N H Z H VN' N N N hn-n'N . A>, I NH n*n
N H a H Vn , N N NH o
include:
[0139] The compounds disclosed herein, in some embodiments, are used in different enriched
3 11 13 14 isotopic forms, e.g., enriched in the content of Η, H, C, C and/or C. In one particular embodiment, the compound is deuterated in at least one position. Such deuterated forms can be made by the procedure described in U.S. Patent Nos. 5,846,514 and 6,334,997. As described in U.S. Patent Nos. 5,846,514 and 6,334,997, deuteration can improve the metabolic stability and or efficacy, thus increasing the duration of action of drugs.
[0140] Unless otherwise stated, structures depicted herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of the present disclosure.
[0141] The compounds of the present disclosure optionally contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds. For example, the compounds may be
3 125 labeled with isotopes, such as for example, deuterium ( H), tritium ( H), iodine-125 ( I) or carbon-14 (14C). Isotopic substitution with 2H, nC, 13C, 14C, 15C, 12N, 13N, 15N, 16N, 160,17O, 14F, 15F, 16F, 17F, 18F, 33S, 34S, 35S, 36S, 35C1,37C1,79Br, 81Br, 12T are all contemplated. All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
-33[0142] In certain embodiments, the compounds disclosed herein have some or all of the 'H atoms replaced with H atoms. The methods of synthesis for deuterium-containing compounds are known in the art and include, by way of non-limiting example only, the following synthetic methods. Deuterium substituted compounds are synthesized using various methods such as described in:
Dean, Dennis C.; Editor. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000; 6(10)] 2000, 110 pp; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.
[0143] Deuterated starting materials are readily available and are subjected to the synthetic methods described herein to provide for the synthesis of deuterium-containing compounds. Large numbers of deuterium-containing reagents and building blocks are available commercially from chemical vendors, such as Aldrich Chemical Co.
[0144] Compounds of the present invention also include crystalline and amorphous forms of those compounds, pharmaceutically acceptable salts, and active metabolites of these compounds having the same type of activity, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof.
[0145] The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0146] The phrase “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable
-34carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
[0147] “Antibody drug conjugate” (“ADC”) can refer to an antibody constmct immune-stimulatory compound conjugate. An ADC can comprise any embodiment as described herein for an antibody construct immune-stimulatory compound conjugate. Therefore, ADC and antibody construct immune-stimulatory compound conjugate can be used interchangeably herein.
[0148] An antigen can elicit an immune response. An antigen can be a protein, polysaccharide, lipid, or glycolipid, which can be recognized by an immune cell, such as a T cell or a B cell. Exposure of immune cells to one or more of these antigens can elicit a rapid cell division and differentiation response resulting in the formation of clones of the exposed T cells and B cells. B cells can differentiate into plasma cells which in turn can produce antibodies which selectively bind to the antigens.
[0149] In cancer, there are four general groups of tumor antigens: (i) viral tumor antigens which can be identical for any viral tumor of this type, (ii) carcinogenic tumor antigens which can be specific for patients and for the tumors, (iii) isoantigens of the transplantation type or tumor-specific transplantation antigens which can be different in all individual types of tumor but can be the same in different tumors caused by the same virus; and (iv) embryonic antigens.
[0150] As a result of the discovery of tumor antigens, tumor antigens have become important in the development of new cancer treatments that can specifically target the cancer. This has led to the development of antibodies directed against these tumor antigens.
[0151] In addition to the development of antibodies against tumor antigens for cancer treatment, antibodies that target immune cells to boost the immune response have also been developed. For example, an anti-CD40 antibody that is a CD40 agonist can be used to activate dendritic cells to enhance the immune response.
-35[0152] Cluster of Differentiation 40 (CD40) is a member of the Tumor Necrosis Factor Receptor (TNF-R) family. CD40 can be a 50 kDa cell surface glycoprotein that can be constitutively expressed in normal cells, such as monocytes, macrophages, B lymphocytes, dendritic cells, endothelial cells, smooth muscle cells, fibroblasts and epithelium, and in tumor cells, including Bcell lymphomas and many types of solid tumors. Expression of CD40 can be increased in antigen presenting cells in response to IL-Ιβρ, IFN-γ, GM-CSF, and LPS induced signaling events.
[0153] Humoral and cellular immune responses can be regulated, in part, by CD40. For example, in the absence of CD40 activation by its cognate binding partner, CD40 Ligand (CD40L), antigen presentation can result in tolerance. However, CD40 activation can ameliorate tolerance. In addition, CD40 activation can positively impact immune responses by enhancing antigen presentation by antigen presenting cells (APC), increasing cytokine and chemokine secretion, stimulating expression of and signaling by co-stimulatory molecules, and activating the cytolytic activity of different types of immune cells. Accordingly, the interaction between CD40 and CD40L can be essential to maintain proper humoral and cellular immune responses.
[0154] The intracellular effects of CD40 and CD40L interaction can include association of the CD40 cytoplasmic domain with TRAFs (TNF-R associated factors), which can lead to the activation of NFkB and Jun/APl pathways. While the response to activation of NFkB and Jun/APl pathways can be cell type-specific, often such activation can lead to increased production and secretion of cytokines, including IL-6, IL-8, IL-12, IL-15; increased production and secretion of chemokines, including MIPla and β and RANTES; and increased expression of cellular adhesion molecules, including ICAM. While the effects of cytokines, chemokines and cellular adhesion molecules can be widespread, such effects can include enhanced survival and activation of T cells.
[0155] In addition to the enhanced immune responses induced by CD40 activation, CD40 activation can also be involved in chemokine- and cytokine-mediated cellular migration and differentiation; activation of immune cells, including monocytes; activation of and increased cytolytic activity of immune cells, including cytolytic T lymphocytes and natural killer cells; induction of CD40-positive tumor cell apoptosis and enhanced immunogenicity of CD40-positive tumors. In addition, CD40 can initiate and enhance immune responses by many different mechanisms, including, inducing antigenpresenting cell maturation and increased expression of costimulatory molecules, increasing production of and secretion of cytokines, and enhancing effector functions.
[0156] CD40 activation can be effective for inducing immune-mediated antitumor responses. For example, CD40 activation reverses host immune tolerance to tumor-specific antigens which leads to enhanced antitumor responses by T cells. Such antitumor activity can also occur in the absence of
-36immune cells. Similarly, antitumor effects can occur in response to anti-CD40 antibody-mediated activation of CD40 and can be independent of antibody-dependent cellular cytotoxicity. In addition to other CD40-mediated mechanisms of antitumor effects, CD40L-stimulation can cause dendritic cell maturation and stimulation. CD40L-stimulated dendritic cells can contribute to the antitumor response. Furthermore, vaccination strategies including CD40 can result in regression of CD40positive and CD40-negative tumors.
[0157] CD40 activating antibodies (e.g., anti-CD40 activating monoclonal antibodies) can be useful for treatment of tumors. This can occur through one or more mechanisms, including cell activation, antigen presentation, production of cytokines and chemokines, amongst others. For example, CD40 antibodies activate dendritic cells, leading to processing and presentation of tumor antigens as well as enhanced immunogenicity of CD40-positive tumor cells. Not only can enhanced immunogenicity result in activation of CD40-positive tumor specific CD4+ and CD8+ T cells, but further antitumor activity can include, recruitment and activation monocytes, enhanced cytolytic activity of cytotoxic lymphocytes and natural killer cells as well as induction of apoptosis or by stimulation of a humoral response so as to directly target tumor cells. In addition, tumor cell debris, including tumor-specific antigens, can be presented to other cells of the immune system by CD40-activated antigen presenting cells.
[0158] Since CD40 can be important in an immune response, there is a need for enhanced CD40 meditated signaling events to provide reliable and rapid treatment options to patients suffering from diseases which may be ameliorated by treatment with CD40-targeted therapeutic strategies.
[0159] The HER2/neu (human epidermal growth factor receptor 2/receptor tyrosine-protein kinase erbB-2) is part of the human epidermal growth factor family. Overexpression of this protein has been shown to play an important role in the progression of cancer, for example, breast cancer. The HER2/neu protein functions as a receptor tyrosine kinase and autophosphorylates upon dimerization with binding partners. HER2/neu can activate several signaling pathways including, for example, mitogen-activated protein kinase, phosphoinositide 3-kinase, phospholipase Cy, protein kinase C, and signal transducer and activator of transcription (STAT). Several compounds have been developed to inhibit HER2/neu including for example, the monoclonal antibody trastuzumab and the monoclonal antibody pertuzumab.
[0160] Immune-stimulatory molecular motifs, such as Pathogen-Associated Molecular Pattern molecules, (PAMPs) can be recognized by receptors of the innate immune system, such as Toll-like receptors (TLRs), Nod-like receptors, C-type lectins, and RIG-I-like receptors. These receptors can be transmembrane and intra-endosomal proteins which can prime activation of the immune system
-37in response to infectious agents such as pathogens. Similar to other protein families, TLRs can have many isoforms, including TLR4, TLR7 and TLR8. Several agonists targeting activation of different TLRs can be used in various immunotherapies, including vaccine adjuvants and in cancer immunotherapies. TLR agonists can range from simple molecules to complex macromolecules. Likewise, the sizes of TLR agonists can range from small to large. TLR agonists can be synthetic or biosynthetic agonists. TLR agonists can also be PAMPs. Additional immune-stimulatory compounds, such as cytosolic DNA and unique bacterial nucleic acids called cyclic dinucleotides, can be recognized by Interferon Regulatory Factor (IRF) or stimulator of interferon genes (STING), which can act a cytosolic DNA sensor. Compounds recognized by Interferon Regulatory Factor (IRF) can play a role in immunoregulation by TLRs and other pattern recognition receptors.
[0161] Imiquimod, a synthetic TLR7 agonist, is currently approved for human therapeutic applications. Contained in a cream and marketed under the brand name Aldara, imiquimod serves as a topical treatment for a variety of indications with immune components, such as, actinic keratosis, genital warts, and basal cell carcinomas. In addition, imiquimod is indicated as a candidate adjuvant for enhancing adaptive immune responses when applied topically at an immunization site.
[0162] Another type of immune stimulatory molecular motif, damage-associated molecular pattern molecules (DAMPs), can initiate and maintain an immune response occurring as part of the noninfectious inflammatory response. DAMPs can be specially localized proteins that, when detected by the immune system in a location other than where DAMPs should be located, activate the immune system. Often, DAMPs can be nuclear or cytosolic proteins and upon release from the nucleus or cytosol, DAMP proteins can become denatured through oxidation. Examples of DAMP proteins can include chromatin-associated protein high-mobility group box 1 (HMGB1), SI 00 molecules of the calcium modulated family of proteins and glycans, such as hyaluronan fragments, and glycan conjugates. DAMPs can also be nucleic acids, such as DNA, when released from tumor cells following apoptosis or necrosis. Examples of additional DAMP nucleic acids can include RNA and purine metabolites, such as ATP, adenosine and uric acid, present outside of the nucleus or mitochondria.
[0163] Therapeutic application of DAMPs can focus on indications with an immune component, such as arthritis, cancer, ischemia-reperfusion injury, myocardial infarction and stroke. In these indications, the mechanism of action for DAMP therapeutic effects can include the prevention of DAMP release using therapeutic strategies, such as proapoptotic interventions, platinum and ethyl pyruvate, extracellular neutralization or blockade of DAMP release or signaling using therapeutic strategies such as anti-HMGBl, rasburiaspect and sRAGE, as well as direct or indirect blockade of
-38DAMP receptors, and downstream signaling events, using therapeutic strategies such as RAGE small molecule antagonists; TLR4 antagonists and antibodies to DAMP-R.
[0164] Additionally, the immune response elicited by TLR agonists can further be enhanced when co-administered with a CD40-agonist antibody. For example, co-administration of a TLR agonist such as poly IC:LC with a CD40-agonist antibody can synergize to stimulate a greater CD8+ T cell response than either agonist alone.
[0165] However, therapeutic use of PAMPs and DAMPs or other mechanisms of intervention can be limited because systemic activation of PAMP and DAMP signaling pathways can have lifethreatening consequences due to cytokine syndrome-induced or cytokine storm-induced toxic shock syndrome. Accordingly, there is a critical need for therapeutic, clinically relevant targeted delivery of PAMP and DAMP agonists for safe and effective strategies to enhance immune responses. The presently described conjugate can be utilized as a safe and effective strategy to enhance immune responses. A conjugate can comprise an antibody construct and an immune-stimulatory compound.
Antibody Construct [0166] An antibody construct can comprise an antigen binding domain. An antigen binding domain can be a domain that can specifically bind to an antigen. An antigen binding domain can be an antigen-binding portion of an antibody or an antibody fragment. An antigen binding domain can be one or more fragments of an antibody that can retain the ability to specifically bind to an antigen. An antigen binding domain can be any antigen binding fragment. An antigen binding domain can recognize a single antigen. An antigen binding domain can recognize, for example, two, three, four, five, six, seven, eight, nine, ten, or more antigens. An antibody construct can contain, for example, two, three, four, five, six, seven, eight, nine, ten, or more antigen binding domains. An antibody construct can contain two antigen binding domains in which each antigen binding domain can recognize the same antigen. An antibody construct can contain two antigen binding domains in which each antigen binding domain can recognize different antigens. An antigen binding domain can be in a scaffold, in which a scaffold is a supporting framework for the antigen binding domain. An antigen binding domain can be in a non-antibody scaffold. An antigen binding domain can be in an antibody scaffold. An antibody construct can comprise an antigen binding domain in a scaffold. The antibody construct can comprise a Fc fusion protein product. In some embodiments, the antibody construct is a Fc fusion protein product.
[0167] The antigen binding domain of an antibody construct can be selected from any domain that binds the antigen including, but not limited to, from a monoclonal antibody, a polyclonal antibody, a
-39recombinant antibody, or a functional fragment thereof, for example, a heavy chain variable domain (Vh) and a light chain variable domain (Vl), a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor, or a recombinant T cell receptor. The antigen binding domain of an antibody construct can be at least 80% homologous to an antigen binding domain selected from, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or a functional fragment thereof, for example, a heavy chain variable domain (Vh) and a light chain variable domain (Vl), a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor, or a recombinant T cell receptor.
[0168] An antigen binding domain of an antibody construct, for example an antigen binding domain from a monoclonal antibody, can comprise a light chain and a heavy chain. In one aspect, the monoclonal antibody binds to CD40 and comprises the light chain of an anti-CD40 antibody and the heavy chain of an anti-CD40 antibody, which bind a CD40 antigen. In another aspect, the monoclonal antibody binds to a tumor antigen and comprises the light chain of a tumor antigen antibody and the heavy chain of a tumor antigen antibody, which bind the tumor antigen.
[0169] An antibody construct can be an antibody. An antibody can consist of two identical light protein chains and two identical heavy protein chains, all held together covalently by precisely located disulfide linkages. The N-terminal regions of the light and heavy chains together can form the antigen recognition site of an antibody. Structurally, various functions of an antibody can be confined to discrete protein domains (i.e., regions). The sites that can recognize and can bind antigen can consist of three complementarity determining regions (CDRs) that can lie within the variable heavy chain region and variable light chain region at the N-terminal end of the heavy chain and the light chain. The constant domains can provide the general framework of the antibody and may not be involved directly in binding the antibody to an antigen, but can be involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity, and can bind Fc receptors.
[0170] The domains of natural light and heavy chains can have the same general structures, and each domain can comprise four framework regions, whose sequences can be somewhat conserved, connected by three hyper-variable regions or CDRs. The four framework regions can largely adopt a β-sheet conformation and the CDRs can form loops connecting, and in some aspects forming part of, the β -sheet structure. The CDRs in each chain can be held in close proximity by the framework
-40regions and, with the CDRs from the other chain, can contribute to the formation of the antigen binding site.
[0171] An antibody of an antibody construct can include an antibody of any type, which can be assigned to different classes of immunoglobins, e.g., IgA, IgD, IgE, IgG, and IgM. Several different classes can be further divided into isotypes, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. An antibody can further comprise a light chain and a heavy chain, often more than one chain. The heavy-chain constant regions (Fc) that corresponds to the different classes of immunoglobulins can be α, δ, ε, γ, and μ, respectively. The light chains can be one of either kappa or κ and lambda or λ, based on the amino acid sequences of the constant domains. The Fc region can contain an Fc domain. An Fc receptor can bind an Fc domain. Antibody constructs can also include any fragment or recombinant forms thereof, including but not limited to, single chain variable fragments (scFvs), ‘T-bodies’, anti-calins, centyrins, affibodies, domain antibodies, or peptibodies.
[0172] An antibody can comprise an antigen binding domain, which can refer to a portion of an antibody comprising the antigen recognition portion, i.e., an antigenic determining variable region of an antibody sufficient to confer recognition of the antigen and binding of the antigen recognition portion to a target, such as an antigen, i.e., the epitope. Examples of antibody binding domains can include, but are not limited to, Fab, scFv, variable Fv fragment, and other antibody fragments, combinations of fragments or types of fragments known or knowable to one of ordinary skill in the art.
[0173] An antibody construct can comprise an antigen binding domain of an antibody. An antigen binding domain of an antibody can comprise one or more light chain (LC) CDRs and one or more heavy chain (HC) CDRs. For example, an antibody binding domain of an antibody can comprise one or more of the following: a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), or a light chain complementary determining region 3 (LC CDR3). For another example, an antibody binding domain can comprise one or more of the following: a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), or a heavy chain complementary determining region 3 (HC CDR3). As an additional example, an antibody binding domain of an antibody can comprise one or more of the following: LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3.
[0174] An antibody construct can comprise an antibody fragment. An antibody fragment can include (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl and Chi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at
-41the hinge region; and (iii) a Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody. Although the two domains of the Fv fragment, Vl and Vh, can be coded for by separate genes, they can be linked by a synthetic linker to be made as a single protein chain in which the Vl and Vh regions pair to form monovalent molecules.
[0175] F(ab')2 and Fab' moieties can be produced by treating immunoglobulin (e.g., monoclonal antibody) with a protease such as pepsin and papain, and can include an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. The Fab fragment can also contain the constant domain of the light chain and the first constant domain (Chi) of the heavy chain. Fab' fragments can differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain Chi domain including one or more cysteine(s) from the antibody hinge region.
[0176] An Fv can be the minimum antibody fragment which contains a complete antigenrecognition and antigen-binding site. This region can consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. In this configuration the three hypervariable regions of each variable domain can interact to define an antigen-binding site on the surface of the Vh-Vl dimer. A single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) can recognize and bind antigen, although the binding can be at a lower affinity than the affinity of the entire binding site.
[0177] An antibody used herein can be “humanized.” Humanized forms of non-human (e.g., murine) antibodies can be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of antibodies), which can contain minimal sequences derived from non-human immunoglobulin. In general, the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
[0178] An antibody described herein can be a human antibody. As used herein, “human antibodies” can include antibodies having, for example, the amino acid sequence of a human immunoglobulin and can include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulins. Human antibodies can be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin -42genes. Completely human antibodies that recognize a selected epitope can be generated using guided selection. In this approach, a selected non-human monoclonal antibody, e.g., a mouse antibody, can be used to guide the selection of a completely human antibody recognizing the same epitope.
[0179] An antibody described herein can be a bispecific antibody or a dual variable domain antibody (DVD). Bispecific and DVD antibodies can be monoclonal, often human or humanized, antibodies that can have binding specificities for at least two different antigens.
[0180] An antibody described herein can be a derivatized antibody. For example, derivatized antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein.
[0181] An antibody described herein can have a sequence that has been modified to alter at least one constant region-mediated biological effector function relative to the corresponding wild type sequence. For example, in some embodiments, the antibody can be modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g., reduced binding to the Fc receptor (FcR). FcR binding can be reduced by, for example, mutating the immunoglobulin constant region segment of the antibody at particular regions necessary for FcR interactions.
[0182] An antibody described herein can be modified to acquire or improve at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g., to enhance FcyR interactions. For example, an antibody with a constant region that binds FcyRIIA, FcyRIIB and/or FcyRIIIA with greater affinity than the corresponding wild type constant region can be produced according to the methods described herein.
[0183] An antibody described herein can bind to tumor cells, such as an antibody against a cell surface receptor or a tumor antigen. An antibody described herein can bind to CD40, such as an antibody that can be a CD40 agonist and bind to CD40.
[0184] As used herein, the abbreviations for the natural 1-enantiomeric amino acids are conventional and can be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, lie); leucine (F, Feu); lysine (K, Fys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X can indicate any amino acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H), lysine (K), or arginine (R).
-43[0185] An antibody construct can comprise an anti-CD40 antibody. An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be expressed from a DNA sequence comprising
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGC
GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAGCAGAAAC
CAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCA
TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA
ACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCGG
CGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCC
CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 1). A light chain of an anti-CD40 antibody can be expressed from DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 1. A variable region of a light chain of an anti-CD40 antibody can be expressed from a DNA sequence comprising
GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCAC
CATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAGCAGAAAC CAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCA TCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA ACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCACTTTCGG CGGAGGGACCAAGGTGGAGATCAA (SEQ ID NO: 3). A variable region of a light chain of an anti-CD40 antibody can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 3. Additionally, anti-CD40 antibodies expressed from SEQ ID NO:
1, or expressed from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 1 can have a dissociation constant (Ka) for CD40 that is less than lOnM. Anti-CD40 antibodies expressed from SEQ ID NO: 1, or expressed from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 1 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100
-44pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. The anti-CD40 light chain can be expressed with any anti-CD40 heavy chain or fragment thereof. The anti-CD40 light chain can also expressed with any anti-CD40 heavy chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. [0186] A light chain of an anti-CD40 antibody can comprise an amino acid sequence
MRFPAQFFGFLFFWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWFAWYQQKPG KAPNLFIYTASTFQSGVPSRFSGSGSGTDFTLTISSFQPEDFATYYCQQANIFPFTFGGGTKVE IKRTVAAPSVFIFPPSDEQFKSGTASVVCFFNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSFSSTFTFSKADYEKHKVYACEVTHQGFSSPVTKSFNRGEC (SEQ ID NO: 4). A light chain of an anti-CD40 antibody can comprise an amino sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 4. A variable region of a light chain of an anti-CD40 antibody can comprise an amino acid sequence
DIQMTQSPSSVSASVGDRVTITCRASQGIYSWFAWYQQKPGKAPNFFIYTASTFQSGVPSRF SGSGSGTDFTFTISSFQPEDFATYYCQQANIFPFTFGGGTKVEIK (SEQ ID NO: 6). A variable region of a light chain of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 6. Additionally, anti-CD40 antibodies comprising SEQ ID NO: 4, or comprising an amino acid sequence with greater than 70% homology to SEQ ID NO: 4 can have a dissociation constant (Ka) for CD40 that is less than lOnM. Anti-CD40 antibodies comprising SEQ ID NO: 4, or comprising an amino acid sequence with greater than 70% homology to SEQ ID NO: 4 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. The anti-CD40 light chain can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 light chain can be combined with any anti-CD40 heavy chain or fragment thereof. The anti-CD40 light chain can also be combined with any anti-CD40 heavy chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0187] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40
-45antibody can be SBT-040 VL-Ck. SBT-040 VL-Ck can comprise an amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26). SBT-040 VL-Ck can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 26.
[0188] A light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QGIYSW (SEQ ID NO: 27). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence TAS (SEQ ID NO: 28). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QQANIFPLT (SEQ ID NO: 29). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 27. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 28. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 29.
[0189] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40 antibody can be an IgGl isotype. A heavy chain of an anti-CD40 antibody can be Dacetuzumab. Dacetuzumab can comprise an amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGTSY
NQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK (SEQ ID NO: 38). Dacetuzumab can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than 95% or greater than 99% homology to SEQ ID NO: 38. A heavy chain of an anti-46CD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GYSFTGYY (SEQ ID NO: 39). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence VIPNAGGT (SEQ ID NO: 40). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence AREGIYW (SEQ ID NO: 41). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 39. A heavy chain CDR of an antiCD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 40. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 41. The two-dimensional structure of the dacetuzumab heavy chain is shown in FIGURE 16.
[0190] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be dacetuzumab. Dacetuzumab can comprise an amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQKPGKAPKLLIYTVSNRFSG VPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42). Dacetuzumab can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 42. A light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QSLVHSNGNTF (SEQ ID NO: 43). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence TVS (SEQ ID NO: 44). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence SQTTHVPWT (SEQ ID NO: 45). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 43. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 44. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%,
-47greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 45. The twodimensional structure of the Dacetuzumab light chain is shown in FIGURE 17.
[0191] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40 antibody can be an IgG4 isotype. A heavy chain of an anti-CD40 antibody can be Bleselumab. Bleselumab can comprise an amino acid sequence
QLQLQESGPGLLKPSETLSLTCTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNP SLKSRVTISVDTSKNQF SLKLS S VT AADT AVYYCTRP VVRYFGWFDPWGQGTL VTVS S AST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK (SEQ ID NO: 46). Bleselumab can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 46. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GGSISSPGYY (SEQ ID NO: 47). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence IYKSGST (SEQ ID NO: 48). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence TRPVVRYFGWFDP (SEQ ID NO: 49). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 47. A heavy chain CDR of an antiCD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 48. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 49. The two-dimensional structure of the bleselumab heavy chain is shown in FIGURE 18.
[0192] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be Bleselumab. Bleselumab can comprise an amino acid sequence
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASNLESGVPSRFS
-48GSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50). Bleselumab can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 50. A light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QGISSA (SEQ ID NO: 51). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence DAS (SEQ ID NO: 52). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QQFNSYPT (SEQ ID NO: 53). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 51. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 52. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 53. The two-dimensional structure of the bleselumab light chain is shown in FIGURE 19.
[0193] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40 antibody can be an IgGl isotype. Lucatumumab can comprise an amino acid sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNRYH ADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSSA STKGPSVFPLAPASKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54). Lucatumumab can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 54. A heavy chain of an antiCD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GFTFSSYG (SEQ ID NO: 55). A heavy chain of an anti-CD40
-49antibody can comprise a CDR with an amino acid sequence ISYEESNR (SEQ ID NO: 56). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence ARDGGIAAPGPDY (SEQ ID NO: 57). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 55. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 56. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 57.
The two-dimensional structure of the lucatumumab heavy chain is shown in FIGURE 20.
[0194] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be Lucatumumab. Lucatumumab can comprise an amino acid sequence DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRASGV PDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58). Lucatumumab can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 59. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence LGS (SEQ ID NO: 60). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence MQARQTPFT (SEQ ID NO: 61). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 59. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 60. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 61.
The two-dimensional structure of the lucatumumab light chain is shown in FIGURE 21.
[0195] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40
-50antibody can be an IgGl isotype. A heavy chain of an anti-CD40 antibody can be ADC-1013. ADC1013 can comprise an amino acid sequence
EVQLLESGGGLVQPGGSLRLSC AASGFTF STYGMHWVRQAPGKGLEWLS YISGGS S YIF YA DSVRGRFTISRDNSENALYLQMNSLRAEDTAVYYCARILRGGSGMDLWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCNAVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO: 62). ADC-1013 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 62. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GFTFSTYG (SEQ ID NO: 63). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence ISGGSSYI (SEQ ID NO: 64). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence ARILRGGSGMDL (SEQ ID NO: 65). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 63. A heavy chain CDR of an antiCD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 64. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 65. The two-dimensional structure of the ADC-1013 heavy chain is shown in FIGURE 22.
[0196] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be ADC-1013. ADC-1013 can comprise an amino acid sequence
QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVYWYQQLPGTAPKLLIYGNINRPSGVPDR FSGSKSGTSASLAISGLRSEDEADYYCAAWDKSISGLVFGGGTKLTVLGQPKAAPSVTLFPP SSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 66). ADC-1013 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%,
-51greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 66. A light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence SSNIGAGYN (SEQ ID NO: 67). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GNI (SEQ ID NO: 68). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence AAWDKSISGLV (SEQ ID NO: 69). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 67. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 68. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 69. The twodimensional structure of the ADC-1013 light chain is shown in FIGURE 23.
[0197] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40 antibody can be the humanized rabbit antibody APX005. APX005 can comprise an amino acid sequence
QVQLVESGGGVVQPGRSLRLSCAASGFSFSSTYVCWVRQAPGKGLEWIACIYTGDGTNYSA SWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYFCARPDITYGFAINFWGPGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70). APX005 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 70. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GFSFSSTY (SEQ ID NO: 71). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence IYTGDGTN (SEQ ID NO: 72). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence ARPDITYGFAINFW (SEQ ID NO: 73). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
-52with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 71. A heavy chain CDR of an antiCD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 72. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 73. The two-dimensional structure of the APX005 heavy chain is shown in FIGURE 24.
[0198] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be the humanized rabbit antibody APX005. APX005 can comprise an amino acid sequence
DIQMTQSPSSLSASVGDRVTIKCQASQSISSRLAWYQQKPGKPPKLLIYRASTLASGVPSRFS GSGSGTDFTLTISSLQPEDVATYYCQCTGYGISWPIGGGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 74). APX005 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 74. A light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QSISSR (SEQ ID NO: 75). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence RAS (SEQ ID NO: 76). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QCTGYGISWP (SEQ ID NO: 77). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 75. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 76. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 77. The two-dimensional structure of the APX005 light chain is shown in FIGURE 25.
[0199] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40
-53antibody can be Chi Lob 7/4. Chi Lob 7/4 can comprise an amino acid sequence
EVQLQQSGPDLVKPGASVKISCKTSGYTFTEYIMHWVKQSHGKSLEWIGGIIPNNGGTSYNQ KFKDKATMTVDKSSSTGYMELRSLTSEDSAVYYCTRREVYGRNYYALDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78). Chi Lob 7/4 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 78. A heavy chain of an antiCD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GYTFTEYI (SEQ ID NO: 79). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence IIPNNGGT (SEQ ID NO: 80). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence
TRREVYGRNYYALDY (SEQ ID NO: 81). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 79. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 80. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 81.
The two-dimensional structure of the Chi Lob 7/4 heavy chain is shown in FIGURE 26.
[0200] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be Chi Lob 7/4. Chi Lob 7/4 can comprise an amino acid sequence
DIQMTQTTSSLSASLGDRVTITCSASQGINNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFS GSGSGTDYSLTISNLEPEDIATYYCQQYSNLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82). Chi Lob 7/4 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 82. A light chain of an
-54anti-CD40 antibody can comprise a CDR. A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QGINNY (SEQ ID NO: 83). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence YTS (SEQ ID NO: 84). A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence QQYSNLPYT (SEQ ID NO: 85). A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 83. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 84. A light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 85. The two-dimensional structure of the Chi Lob 7/4 light chain is shown in FIGURE 27.
[0201] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40 antibody can be an IgGl isotype. A heavy chain of an anti-CD40 antibody can be SBT-040-G1WT. SBT-040-G1WT can be expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCA
GGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTC
TCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAAC
TATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAG
CCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGC
CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
-55TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCCCCGGGTAAATGA (SEQ ID NO: 8). SBT-040-G1WT can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 8. A variable region of SBT-040-G1WT can be expressed from a DNA sequence comprising
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGG TCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAG GCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAA ACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCAC AGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGA GAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 13). A variable region of SBT-040G1WT can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40 antibodies comprising SBT-040-G1WT expressed from SEQ ID NO: 8, or expressed from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 8 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1WT expressed from DNA sequence comprising SEQ ID NO: 8, or comprising greater than 70% homology to SEQ ID NO: 8 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1WT can be expressed with any anti-CD40 light chain or fragment thereof. SBT-040-G1WT can also be expressed with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these
-56same concepts could apply to antibody constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0202] SBT-040-G1WT can comprise an amino acid sequence
MDWTWRIEFEVAAATGAHSQVQEVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ
APGQGFEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMEFNRFRSDDTAVYYCAR
DQPFGYCTNGVCSYFDYWGQGTFVTVSSASTKGPSVFPFAPCSRSTSESTAAFGCFVKDYF
PEPVTVSWNSGAFTSGVHTFPAVFQSSGFYSFSSVVTVPSSNFGTQTYTCNVDHKPSNTKV
DKTVEPKSCDKTHTCPPCPAPEFFGGPSVFFFPPKPKDTFMISRTPEVTCVVVDVSHEDPEV
I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPI
EKTISKAKGQPREPQVYTFPPSREEMTKNQVSFTCFVKGFYPSDIAVEWESNGQPENNYKTT
PPVFDSDGSFFFYSKFTVDKSRWQQGNVFSCSVMHEAFHNHYTQKSFSFSPGK (SEQ ID
NO: 15). SBT-040-G1WT can comprise an amino acid sequence with greater than 70%, greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 15. SBT-040-G1WT can comprise an amino acid sequence
QVQFVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGFEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMEFNRFRSDDTAVYYCARDQPFGYCTNGVCSYFDYWG QGTFVTVSS (SEQ ID NO: 20). A variable region of SBT-040-G1WT can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20. Additionally, anti-CD40 antibodies comprising SBT-040-G1WT with SEQ ID NO: 15 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 15 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1WT with SEQ ID NO: 15 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 15 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1WT can be purified. SBT-040-G1WT can be combined with any antiCD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The antiCD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising antiCD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0203] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy chain of an anti-CD40 antibody can be an IgGl isotype. A heavy chain of an anti-CD40 antibody can be SBT-040 VH-57hlgGl wt. SBT-040 VH-hlgGl wt can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to an amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22). A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence GYTFTYY (SEQ ID NO: 23). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence INPDSGGT (SEQ ID NO: 24). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence
ARDQPLGYCTNGVCSYFDY (SEQ ID NO: 25). A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 23. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 24. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 25.
[0204] A heavy chain of an anti-CD40 antibody can be an IgG2 isotype. A heavy chain of an antiCD40 antibody can be SBT-040-G2. SBT-040-G2 be expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCA
GGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTC
TCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAAC
TATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAG
CCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCA
-58GGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGC
CCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC
CCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAG
ACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTG
TGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCT
CCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAG GTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA (SEQ ID NO: 7). SBT-040-G2 can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 7. A variable region of SBT040-G2 can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G2 can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40 antibodies comprising SBT-040-G2 expressed from SEQ ID NO: 7, or expressed from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 7 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G2 expressed from DNA sequence comprising SEQ ID NO: 7, or comprising greater than 70% homology to SEQ ID NO: 7 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G2 can be expressed with any anti-CD40 light chain or fragment thereof. SBT040-G2 can also be expressed with any anti-CD40 light chain or fragment thereof to form an antiCD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an
-59antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0205] SBT-040-G2 can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14). SBT-040-G2 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 14. SBT-040-G1WT can comprise an amino acid sequence SEQ ID NO: 20. A variable region of SBT-040-G2 can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20. Additionally, anti-CD40 antibodies comprising SBT-040-G2 with SEQ ID NO: 14 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 14 can have a dissociation constant (Kd) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G2 with SEQ ID NO: 14 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 14 can have a dissociation constant (Kd) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G2 can be purified. SBT-040-G2 can be combined with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0206] An antibody construct can comprise an antibody with modifications occurring at least at one amino acid residue. Modifications can be substitutions, additions, mutations, deletions, or the like.
An antibody modification can be an insertion of an unnatural amino acid.
-60[0207] An antibody construct can comprise a light chain of an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to the natural or original amino acid sequence. An antibody construct can comprise a heavy chain of an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to the natural or original amino acid sequence. A heavy chain can be the heavy chain of an anti-CD40 antibody which can bind to the CD40 antigen.
[0208] An antibody construct can be an IgGl isotype. An antibody construct can be an IgG2 isotype. An antibody construct can be an IgG3 isotype. An antibody construct can be an IgG4 isotype. An antibody construct can be of a hybrid isotype comprising constant regions from two or more isotypes. An antibody construct can be an anti-CD40 antibody, in which the anti-CD40 antibody can be a monoclonal human antibody comprising a wild-type sequence of an IgGl isoform, in particular, at an Fc region of the antibody.
[0209] Additional anti-CD40 antibody sequences that can be used in the antibody construct can comprise any sequence as shown below in TABLE 1 or combination thereof:
TABLE 1
Description of Sequence SEQ ID NO: Sequence
Heavy Chain DNA sequence of antibody 3.1.1 86 ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTG TCCATGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCC TGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT AGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGA GTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAG ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATG CGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGT ATTACTGTGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACT ACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCT CAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCA GGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACC AGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAG
ACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGA CAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGC ACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGT GGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAG TTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAG GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGG CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGC CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATG ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA CTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA GAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Heavy Chain protein sequence of Antibody 3.1.1 87 MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGMHWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNA LYLQMNSLRVEDTAVYYCVRRGHQLVLGYYYYNGLDVWGQGTTVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK
Light Chain DNA sequence of Antibody 3.1.1 88 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG GATCCAGTGGGGATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGT CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCT AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA CTGAAAATCAGCAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTG GAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGGCTTCAACAGGGGAGAGTGTTAG
Light Chain protein sequence of Antibody 3.1.1 89 MRLPAQLLGLLMLWVSGSSGDIVLTQSPLSLPVTPGEPASISCRSSQSLLYS NGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRL EAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable Domain of Heavy Chain DNA Sequence of Antibody 3.1.1 90 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATGCGCTGTA TCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTG TGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGG TCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Mature Variable Domain of Heavy Chain Protein Sequence of Antibody 3.1.1 91 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AVISKDGGNKYHADSVKGRFTISRDNSKNALYLQMNSLRVEDTAVYYCV RRGHQLVLGYYYYNGLDVWGQGTTVTVSS
Mature Variable Domain of Light Chain DNA Sequence of Antibody 3.1.1 92 GATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTA CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG CAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Mature Variable Domain of Light Chain Protein 93 DIVLTQSPLSLPVTPGEPAAISCRSSQSLLYSNGYNFLDWYLQKPGQSPQL LIYLGSNRASGVPDPYSGSGSGTDFTLKISRLEAEDVGVYYCMQALQTPR TFGQGTKVEIK
Sequence of Antibody 3.1.1
Heavy Chain DNA (variable domain 3.1.1H- A78T) 94 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTT CTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGT GTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGT CTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Heavy Chain Protein (variable domain 3.1.1H- A78T) 95 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AVISKDGGNKYHADSVKGRFTISRDNSKNTEYEQMNSERVEDTAVYYCV RRGHQFVFGYYYYNGFDVWGQGTTVTVSS
Heavy Chain DNA (variable domain 3.1.1H- A78T-V88A- V97A) 96 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTA TCTGCAAATGAATAGCCTGAGAGcTGAAGACACGGCTGTGTATTACTG TGCGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGG TCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Heavy Chain Protein (variable domain 3.1.1H- A78T-V88A- V97A) 97 QVQFVESGGGVVQPGRSFRFSCAASGFTFSSYGMHWVRQAPGKGFEWV AVISKDGGNKYHADSVKGRFTISRDNSKNTFYFQMNSFRAEDTAVYYCA RRGHQFVFGYYYYNGFDVWGQGTTVTVSS
Light Chain DNA (variable domain 3.1.1L- L4M-L83V) 98 GATATTGTGaTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTA CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Light Chain 99 DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNFLDWYLQKPGQSPQL
Protein (variable domain 3.1.1L- L4M-L83V) FIYFGSNRASGVPDRFSGSGSGTDFTFKISRVEAEDVGVYYCMQAFQTPR TFGQGTKVEIK
Heavy Chain DNA Sequence for Antibody 7.1.2 100 ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTG TCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGC CTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCA GTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTG GAGTGGGTGGCAGTTATATCAAATGATGGAGATAATAAATACCATGCA GACTCCGTGTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGC ACGCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTA TATTACTGTGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGA TTACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGT CACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC GCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG CGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA CCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCA CCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCC CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGT GCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACG GGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGT TGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCT CCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACC AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG GCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGG CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Heavy Chain Protein 101 MEFGFSWVFFVAFFRGVQCQVQFVESGGGVVQPGRSFRFSCAASGFTFS SYGMHWVRQAPGKGFEWVAVISNDGDNKYHADSVWGRFTISRDNSRST
Sequence for Antibody 7.1.2 FYFQMNSFRAEDTAVYYCARRGMGSSGSRGDYYYYYGFDVWGQGTTV TVSSASTKGPSVFPFAPCSRSTSESTAAFGCFVKDYFPEPVTVSWNSGAFT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFFFPPKPKDTFMISRTPEVTCVVVDVSHE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVFTVVHQDWFNGK EYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTFPPSREEMTKNQVSFTCF VKGFYPSDIAVEWESNGQPENNYKTTPPMFDSDGSFFFYSKFTVDKSRW QQGNVFSCSVMHEAFHNHYTQKSFSFSPGK
Light Chain DNA Sequence for Antibody 7.1.2 102 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG GATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCT
AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA CTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTG GAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G
Light Chain Protein Sequence for Antibody 7.1.2 103 MRFPAQFFGFFMFWVSGSSGDIVMTQSPFSFPVTPGEPASISCRSSQSFFY SNGYNFFDWYFQKPGQSPQFFIYFGSNRASGVPDRFSGSGSGTDFTKISRV EAEDVGVYYCMQAFQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQFKSG TASVVCFFNNFYPREAKVQWKVDNAFQSGNSQESVTEQDSKDSTYSFSS TFTFSKADYEKHKVYACEVTHQGFSSPVTKSFNRGEC
Mature Variable Domain of Heavy Chain DNA Sequence of Antibody 7.1.2 104 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATATCAAATGATGGAGATAATAAATACCATGCAGACTCCGT GTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGCACGCTTTA TCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTG
TGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGATTACTACT ACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT CCTCA
Mature Variable Domain of Heavy Chain Protein Sequence of Antibody 7.1.2 105 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AVISNDGDNKYHADSVWGRFTISRDNSRSTFYFQMNSFRAEDTAVYYCA RRGMGSSGSRGDYYYYYGFDVWGQGTTVTVSS
Mature Variable Domain of Light Chain DNA Sequence of Antibody 7.1.2 106 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTA CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Mature Variable Domain of Light Chain Protein Sequence of Antibody 7.1.2 107 DIVMTQSPFSFPVTPGEPASISCRSSQSFFYSNGYNFFDWYFQKPGQSPQF FIYFGSNRASGVPDRFSGSGSGTDFTFKISRVEAEDVGVYYCMQAFQTPR TFGQGTKVEIK
Heavy Chain DNA Sequence for Antibody 10.8.3 108 ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGCAGCTCCCAGATGG GTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAA GCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATC AGTAGTTACTACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTG GAATGGATTGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCC TCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAG TTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTAT TACTGTGCGAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGG GGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCA GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGAT
CACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATG TTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTC AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACC CCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTG GTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAG TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCAT GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAATGA
Heavy Chain Protein Sequence for Antibody 10.8.3 109 MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSISS YYWIWIRQPAGKGLEWIGRVYTSGSTNYNPSLKSRVTMSVDTSKNQFSL KLS SVTAADTAVYYCARDGLYRGYGMDVWGQGTTVTVS SASTKGPS VF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK
Light Chain DNA Sequence for Antibody 10.8.3 110 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAG GTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGC ATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTAT TAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTA AACTCCTGATTTATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAG GTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGACTGACAG TTTCCCGCTCACTTTCGGCGGCGGGACCAAGGTGGAGATCAAACGAAC TGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG
AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCA GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Light Chain Protein Sequence for Antibody 10.8.3 111 MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQPISS WLAWYQQKPGKAPKLLIYSASGLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQTDSFPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQFKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSIYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable Domain of Heavy Chain DNA Sequence for Antibody 10.8.3 112 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTAC TACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAATGGAT TGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAA GAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGC GAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGGGGCCAAG GGACCACGGTCACCGTCTCCTCA
Mature Variable Domain of Heavy Chain Protein Sequence for Antibody 10.8.3 113 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWIWIRQPAGKGLEWIGRV YTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDGL YRGYGMDVWGQGTTVTVSS
Mature Variable Domain of Light Chain DNA Sequence for Antibody 10.8.3 114 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTATTAGCAGCTGG TTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATT TATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGC AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT GAAGATTTTGCAACTTACTATTGTCAACAGACTGACAGTTTCCCGCTCA CTTTCGGCGGCGGGACCAAGGTGGAGATCAAA
Mature Variable Domain of Light Chain Protein 115 DIQMTQSPSSVSASVGDRVTITCRASQPISSWLAWYQQKPGKAPKLLIYSA SGFQSGVPSRFSGSGSGTDFTFTISSFQPEDFATYYCQQTDSFPFTFGGGTK VEIK
Sequence for Antibody 10.8.3
Heavy Chain DNA Sequence for Antibody 15.1.1 116 ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGG TCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGC CTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAG AAGTTACTACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGG AGTGGATTGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCT CCCTCAAGAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGT TCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTTTATT ACTGTGCGAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACC AGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGG GCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGA GCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCT TCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGT AGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCA AATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC CCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGTCACGAAG ACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA ATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCC CATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCGGGTAAATGA
Heavy Chain Protein Sequence for 117 MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSIRS YYWTWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLK LSSVTAADTAVYYCARKGDYGGNFNYFHQWGQGTLVTVSSASTKGPSV
Antibody 15.1.1 FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGL PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK
Light Chain DNA Sequence for Antibody 15.1.1 118 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG GATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCT CCTACATACTAATGGATACAACTATTTCGATTGGTACCTGCAGAAGCC AGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA CTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGCTCTACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTG GAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G
Light Chain Protein Sequence for Antibody 15.1.1 119 MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSLLH TNGYNYFDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQALQTPYSFGQGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable Domain of Heavy Chain DNA Sequence for Antibody 15.1.1 120 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAAGTTAC TACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGAT TGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAA GAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCT GAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGC GAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACCAGTGGGG
CCAGGGAACCCTGGTCACCGTCTCCTCA
Mature Variable Domain of Heavy Chain Protein Sequence for Antibody 15.1.1 121 QVQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWTWIRQPPGKGLEWIGYI YYSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAADTAVYYCARKGD YGGNFNYFHQWGQGTLVTVSS
Mature Variable Domain of Light Chain DNA Sequence for Antibody 15.1.1 122 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATACTA ATGGATACAACTATTTCGATTGGTACCTGCAGAAGCCAGGGCAGTCTC CACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT ACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
Mature Variable Domain of Light Chain Protein Sequence for Antibody 15.1.1 123 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHTNGYNYFDWYLQKPGQSPQL LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPY SFGQGTKLEIK
Heavy Chain DNA Sequence for Antibody 21.4.1 124 ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGA GCCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAA GCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTT CACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCT TGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGC ACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCA GCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCC GTGTATTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGT GTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCT CCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTG ACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTC TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACC CAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT GGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCC
AGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGT GGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAG CAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCAC CAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGC AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTT CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Heavy Chain Protein Sequence for Antibody 21.4.1 125 MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTF TGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSI STAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEY KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK
Light Chain DNA Sequence for Antibody 21.4.1 126 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAG GTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGC ATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTAT TTACAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA CCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAG GTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG CCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATT TTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACT GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA
ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGT CACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Light Chain Protein Sequence for Antibody 21.4.1 127 MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYS WLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQANIFPLTFGGGTI<VEII<RTVAAPSVFIFPPSDEQLI<SGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable Domain of Heavy Chain DNA Sequence of Antibody 21.4.1 128 AGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCC TCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTAC TATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG GGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTA CATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTG TGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTA CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
Mature Variable Domain of Heavy Chain Protein Sequence of Antibody 21.4.1 129 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEW MGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYY CARDQPLGYCTNGVCSYFDYWGQGTLVTVSS
Mature Variable Domain of Light Chain DNA Sequence of Antibody 21.4.1 130 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGG TTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATC TATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGC AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCT GAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCA CTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
Mature Variable Domain of Light Chain Protein Sequence of Antibody 21.4.1 131 DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYT ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGT KVEIK
Heavy Chain DNA Sequence for Antibody 21.2.1 132 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT GTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATGTCATATGATGGAAGTAGTAAATACTATGCAAACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTA TCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG TGCGAGAGATGGGGGTAAAGCAGTGCCTGGTCCTGACTACTGGGGCC AGGGAATCCTGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence for Antibody 21.2.1 133 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWV AVMSYDGSSKYYANSVKGRFTISRDNSKNTLYLQINSLRAEDTAVYYCA RDGGKAVPGPDYWGQGILVTVSS
Light Chain DNA Sequence for Antibody 21.2.1 134 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGTTCTGTATAGTA ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGTTTT ACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light Chain Protein Sequence for Antibody 21.2.1 135 DIVMTQSPLSLPVTPGEPASISCRSSQSVLYSNGYNYLDWYLQKPGQSPQL LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVLQTPF TFGPGTKVDIK
Heavy Chain DNA Sequence for Antibody 22.1.1 136 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATATCATCTGATGGAGGTAATAAATACTATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTA TCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG TACGAGAAGAGGGACTGGAAAGACTTACTACCACTACTGTGGTATGG ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence for Antibody 22.1.1 137 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWV AVISSDGGNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCT RRGTGKTYYHYCGMDVWGQGTTVTVS S
Light Chain DNA Sequence for Antibody 22.1.1 138 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTA ATGGATATAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC CACACCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGTTCAGGCACTGATTTTACACTGAAAATCAG CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light Chain Protein Sequence for Antibody 22.1.1 139 DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPHL LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR TFGQGTKVEIK
Heavy Chain DNA Sequence for Antibody 23.5.1 140 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTCCAGCCTGGGAGGTC CCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTAACTATGGC ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGC AATTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAA GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGT GCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC GAGACGCGGTCACTACGGGAGGGATTACTACTCCTACTACGGTTTGGA CGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence for Antibody 23.5.1 141 QVQLVESGGGVVQPGRSLRLSCVASGFTFSNYGMHWVRQAPGKGLEWV AIISYDGSNKYYADSVKGRFTISRDNSKNTFYVQMNSFRAEDTAVYYCAR RGHYGRDYYSYYGFDVWGQGTTVTVSS
Light Chain DNA Sequence for Antibody 23.5.1 142 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTA ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light Chain Protein Sequence for Antibody 23.5.1 143 DIVMTQSPFSFPVTPGEPASISCRSSQSFFPGNGYNYFDWYFQKPGQSPQF FIYFGSNRASGVPDRFSGSGSGTDFTFKISRVEAEDVGVYYCMQAFQTPR TFGQGTKVEIK
Heavy Chain 144 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
DNA Sequence for Antibody 23.28.1 CACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTAC TACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGAT TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA GAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCT GAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGC GAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence for Antibody 23.28.1 145 QVQLQESGPGLVKPSDTLSLTCTVSGGSIRGYYWSWIIRQPPGKGLEWIGY IYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGG LYGDYGWFAPWGQGTLVTVSS
Light Chain DNA Sequence for Antibody 23.28.1 146 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGG GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAG CGACTTAGCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCT CATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAG TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGA GCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTGTCGTAGCTTATTC ACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light Chain Protein Sequence for Antibody 23.28.1 147 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSDLAWHQQKPGQAPRLLIYG ASSRATGIPDRFSGSGSGTDFTFTISRFEPEDFAVYYCQHCRSFFTFGPGTK VDIK
Heavy Chain DNA Sequence (variable domain 23.28.1H-D16E) 148 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTAC TACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGAT TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA GAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACOAGTTCTCCCT GAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGC GAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence 149 QVQFQESGPGFVKPSFTFSFTCTVSGGSIRGYYWSWIRQPPGKGFEWIGYI YYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGGL YGDYGWFAPWGQGTLVTVSS
-ΊΊ-
(variable domain 23.28.1H-D16E)
Heavy Chain DNA Sequence of Antibody 23.29.1 150 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT GCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGT GAAGGGCCGATTCACCATCTACAGAGACAATTCCAAGAACACGCTGT ATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACT GTGCGAGACGCGGTCACTACGGGAATAATTACTACTCCTATTACGGTT TGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence for Antibody 23.29.1 151 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWV AVISYDGSNKYYADSVKGRFTIYRDNSKNTLYLQMNSLRAEDTAVYYCA RRGHYGNNYYSYYGLDVWGQGTTVTVSS
Light Chain DNA Sequence for Antibody 23.29.1 152 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTA ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGC AGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGC AGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTA CAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light Chain Protein Sequence for Antibody 23.29.1 153 DIVMTQSPLSLPVTPGEPASISCRSSQSLLPGNGYNYLDWYLQKPGQSPQL LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPRT FGQGTKVEIK
Heavy Chain DNA Sequence for Antibody 24.2.1 154 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTAC TACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGAT TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA GAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCT GAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGC GAGAAGGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy Chain Protein Sequence for Antibody 24.2.1 155 QVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYI YYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGGL YGDYGWFAPWGQGTLVTVSS
Light Chain DNA Sequence for Antibody 24.2.1 156 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGG GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACC TACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGT GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATAGTAGCTTATTCA CTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light Chain Protein Sequence for Antibody 24.2.1 157 ETVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYG ASSRATGIIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSLFTFGPGTK VDIK
Heavy Chain DNA Sequence for Antibody 21.2.1 158 ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTG TCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGC CTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCA GTAGCTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGG AGTGGGTGGCAGTTATGTCATATGATGGAAGTAGTAAATACTATGCAA ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA CGCTGTATCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGT ATTACTGTGCGAGAGATGGGGGTAAAGCAGTGCCTGGTCCTGACTACT GGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC CATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCA CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA CGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCC CAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGA CCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAG ATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAA TGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGAC CCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGT
GGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGA GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGA AAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTG GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC CGGGTAAATGA
Heavy Chain Protein Sequence for Antibody 21.2.1 159 MEFGLSWVFLVALLRGVOCQVQLVESGGGVVQPGRSLRLSCAASGFTFS SYVMHWVRQAPGKGLEWVAVMSYDGSSKYYANSVKGRIFTISRDNSKN TFYFQINSFRAEDTAVYYCARDGGKAVPGPDYWGQGIFVTVSSASTKGP SVFPFAPCSRSTSESTAAFGCFVKDYFPEPVTVSWNSGAFTSGVHTFPAVF QSSGFYSFSSVVTVPSSNTGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFFFPPKPKDTFMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVFTVVHQDWFNGKEYKCKVSNK GFPAPIEKTISKTKGQPREPQVYTFPPSREEMTKNQVSFTCFVKGFYPSDIA VEWESNGQPENNYKTTPPMFDSDGSFFFYSKFTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
Light Chain DNA Sequence for Antibody 21.2.1 160 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG GATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGT TCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCC AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA CTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC ATGCAAGTTTTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTG GATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT CTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
Light Chain Protein Sequence for Antibody 21.2.1 161 MRLPAQLLGLLMLWVSGSSGDIVMTQSPLSLPVTPGEPASISCRSSQSVLY SNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQVLQTPFTFGPGTKVDWRTVAAPSVFIFPPSDEQLKS GTASVVCFFNNFYPREAKVQWKVDNAFQSGNSQESVTEQDSKDSTYSFS STFTFSKADYEKHKVYACEVTHQGFSSPVTKSFNRGEC
[0210] Additional anti-CD40 antibody sequences that can be used in the antibody construct can comprise a sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to any sequence in TABLE 1. [0211] Antibody constructs disclosed herein can be non-natural, designed, and/or engineered. Antibody constructs disclosed herein can be non-natural, designed, and/or engineered scaffolds comprising an antigen binding domain. Antibody constructs disclosed herein can be non-natural, designed, and/or engineered antibodies. Antibody constructs can be monoclonal antibodies.
Antibody constructs can be human antibodies. Antibody constructs can be humanized antibodies. Antibody constructs can be monoclonal humanized antibodies. Antibody constructs can be recombinant antibodies.
[0212] An antigen binding domain of an antibody construct can be selected in order to recognize an antigen. For example, an antigen can be a cell surface marker on a target cell associated with a disease or condition. An antigen can be expressed on an immune cell. An antigen can be a peptide or fragment thereof. An antigen can be expressed on an antigen-presenting cell. An antigen can be expressed on a dendritic cell, a macrophage, or a B cell. An antigen can be a peptide presented in a major histocompatibility complex by cell. As another example, a cell surface marker recognized by the antigen binding domain can include macromolecules associated with viral and bacterial diseases or infections, autoimmune diseases and cancerous diseases. An antigen can be CD40 and an antigen binding domain can recognize a CD40 antigen. An antigen can be a tumor antigen or fragment thereof. A tumor antigen can be any antigen listed on tumor antigen databases, such as TANTIGEN, or peptide databases for T cell-defined tumor antigens, such as the Cancer Immunity Peptide database. A tumor antigen can also be any antigen listed in the review by Chen (Chen, Cancer Immun 2004 [updated 2004 Mar 10; cited 2004 Apr 1]). Note that the ‘antibody’ can recognize the ‘tumor antigen’ or a peptide derived thereof, bound to an MHC molecule. An antigen can have at least 80% homology to or can be CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-F1, B7-H3, B7-DC, BCMA, CS-1, PD-F1, B7-H3, B7-DC, HFDDR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250,
-81prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, pl85HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NYBR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, R0R2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, or Fos-related antigen 1. An antigen binding domain can be capable of recognizing a single antigen. An antigen binding domain can be capable of recognizing two or more different antigens.
[0213] An antibody construct can comprise an antibody heavy chain. A heavy chain can be a heavy chain of an anti-HER2 monoclonal antibody which can bind a HER2 antigen. A heavy chain of an anti-HER2 antibody can be an IgGl isotype. A heavy chain of an anti-HER2 antibody can be SBT050 VH-hlgGl wt (pertuzumab). SBT-050 VH-hlgGl wt can comprise an amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIY NQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30). SBT-050 VH-hlgGl wt can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 30. A heavy chain of an anti-HER2 antibody can comprise a CDR. A heavy chain of an anti-HER2 antibody can comprise a CDR with an amino acid sequence GFTFTDYT (SEQ ID NO: 31). A heavy chain of an anti-HER2 antibody can comprise a CDR with an amino acid sequence VNPNSGGS (SEQ ID NO: 32). A heavy chain of an anti-HER2 antibody can comprise a CDR with an amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 33). A heavy chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than
-8285%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 31. A heavy chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 32. A heavy chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 33. [0214] An antibody construct can comprise an antibody light chain. A light chain can be a light chain of a HER2 monoclonal antibody which can bind a HER2 antigen. A light chain of an antiHER2 antibody can be SBT-050 VL-Ck (pertuzumab). SBT-050 VL-Ck can comprise an amino acid sequence
DIQMTQSPSSFSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKFFIYSASYRYTGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCFFNNFYPREAKVQWKVDNAFQSGNSQESVTEQDSKDSTYSFSSTFTFSKADYE KHKVYACEVTHQGFSSPVTKSFNRGEC (SEQ ID NO: 34). SBT-050 VF-Ck can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 34. A light chain of an anti-HER2 antibody can comprise a CDR. A light chain of an anti-HER2 antibody can comprise a CDR with an amino acid sequence QDVSIG (SEQ ID NO: 35). A light chain of an antiHER2 antibody can comprise a CDR with an amino acid sequence SAS (SEQ ID NO: 36). A light chain of an anti-HER2 antibody can comprise a CDR with an amino acid sequence QQYYIYPYT (SEQ ID NO: 37). A light chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 35. A light chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 36. A light chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 37.
[0215] An antibody construct can comprise an Fc region with an Fc domain. An Fc domain is a structure that can bind to Fc receptors (FcRs). An antibody construct can comprise an Fc domain. Fc domains can be bound by FcRs. An Fc domain can be from an antibody. An Fc domain can be at least 80% homologous to an Fc domain from an antibody. An Fc region can be in a scaffold. An Fc region with an Fc domain can be in an antibody scaffold. An Fc region with an Fc domain can be in
-83a non-antibody scaffold. An antibody construct can comprise an Fc region with an Fc domain in an antibody scaffold. An antibody construct can comprise an Fc region with an Fc domain in a nonantibody scaffold. An Fc domain can be in a scaffold. An Fc domain can be in an antibody scaffold. An Fc domain can be in a non-antibody scaffold. An antibody construct can comprise an Fc domain in an antibody scaffold. An antibody construct can comprise an Fc domain in a non-antibody scaffold. Fc domains of antibodies, including those of the present disclosure, can be bound by FcRs. Fc domains can be a portion of the Fc region of an antibody. FcRs can bind to an Fc domain of an antibody. FcRs can bind to an Fc domain of an antibody bound to an antigen. FcRs can be organized into classes (e.g, gamma (γ), alpha (a) and epsilon (ε)) based on the class of antibody that the FcR recognizes. The FcaR class can bind to IgA and includes several isoforms, FcaRI (CD89) and FcapR. The FcyR class can bind to IgG and includes several isoforms, FcyRI (CD64), FcyRIIA (CD32a), Fc/RIIB (CD32b), FcyRIIIA (CD 16a), and FcyRIIIB (CD 16b). An FcyRIIIA (CD 16a) can be an FcyRIIIA (CD16a) F158 variant. An FcyRIIIA (CD16a) can be an FcyRIIIA (CD16a) V158 variant. Each FcyR isoform can differ in affinity to the Fc region of the IgG antibody. For example, FcyRI can bind to IgG with greater affinity than FcyRII or FcyRIII. The affinity of a particular FcyR isoform to IgG can be controlled, in part, by a glycan (e.g., oligosaccharide) at position CH2 84.4 of the IgG antibody. For example, fucose containing CH2 84.4 glycans can reduce IgG affinity for FcyRIIIA. In addition, GO glucans can have increased affinity for FcyRIIIA due to the lack of galactose and terminal GlcNAc moiety.
[0216] Binding of an Fc domain to an FcR can enhance an immune response. FcR-mediated signaling that can result from an Fc region binding to an FcR can lead to the maturation of immune cells. FcR-mediated signaling that can result from an Fc domain binding to an FcR can lead to the maturation of dendritic cells. FcR-mediated signaling that can result from an Fc domain binding to an FcR can lead to more efficient immune cell antigen uptake and processing. FcR-mediated signaling that can result from an Fc region binding to an FcR can lead to more efficient dendritic cell antigen uptake and processing. FcR-mediated signaling that can result from an Fc region binding to an FcR can increase antigen presentation. FcR-mediated signaling that can result from an Fc region binding to an FcR can increase antigen presentation by immune cells. FcR-mediated signaling that can result from an Fc region binding to an FcR can increase antigen presentation by antigen presenting cells. FcR-mediated signaling that can result from an Fc domain binding to an FcR can increase antigen presentation by dendritic cells. FcR-mediated signaling that can result from an Fc domain binding to an FcR can promote the expansion and activation of T cells. FcR-mediated signaling that can result from an Fc domain binding to an FcR can promote the expansion and
-84activation of CD8+ T cells. FcR-mediated signaling that can result from an Fc domain binding to an FcR can influence immune cell regulation of T cell responses. FcR-mediated signaling that can result from an Fc domain binding to an FcR can influence immune cell regulation of T cell responses. FcR-mediated signaling that can result from an Fc domain binding to an FcR can influence dendritic cell regulation of T cell responses. FcR-mediated signaling that can result from an Fc domain binding to an FcR can influence functional polarization of T cells (e.g., polarization can be toward a TH1 cell response).
[0217] The profile of FcRs on a DC can impact the ability of the DC to respond upon stimulation. For example, most DC can express both CD32a and CD32b, which can have opposing effects on IgG-mediated maturation and function of DCs: binding of IgG to CD32a can mature and activate DCs in contrast with CD32b, which can mediate inhibition due to phosphorylation of immunoreceptor tyrosine-based inhibition motif (ITIM), after CD32b binding of IgG. Therefore, the activity of these two receptors can establish a threshold of DC activation. Furthermore, difference in functional avidity of these receptors for IgG can shift their functional balance. Hence, altering the Fc domain binding to FcRs can also shift their functional balance, allowing for manipulation (either enhanced activity or enhanced inhibition) of the DC immune response.
A modification in the amino acid sequence of the antibody construct can alter the recognition and binding of an FcR for the Fc domain. For example, a modification of the amino acid sequence of the Fc domain in an antibody construct can increase the binding affinity and/or avidity of the Fc domain for FcRs. This increase in binding affinity and/or avidity can specific for a type of FcR. However, such modifications can still allow for FcR-mediated signaling. A modification can be a substitution of an amino acid at a residue (e.g., wildtype) for a different amino acid at that residue. For example, a wildtype Fc domain can comprise
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 162), and a modified Fc domain can comprise a substitution of an amino acid in comparison with SEQ ID NO: 162. A modification can permit binding of an FcR to a site on the Fc region of an antibody construct that the FcR may not otherwise bind to. A modification can increase binding affinity of an FcR to the Fc domain of an antibody construct that the FcR may have reduced binding affinity for. A modification can decrease binding
-85affinity of an FcR to a site on the Fc domain of an antibody construct that the FcR may have increased binding affinity for. A modification can increase the subsequent FcR-mediated signaling after Fc binding to an FcR.
[0218] An antibody construct can comprise an Fc region with at least one amino acid change as compared to the sequence of the wild-type Fc region. A wild-type Fc region can comprise SEQ ID NO: 162. An antibody construct can comprise an Fc domain with at least one amino acid change as compared to the sequence of the wild-type Fc domain. A wild-type Fc domain can comprise SEQ ID NO: 162. For example, an antibody construct can comprise an Fc domain with at least one amino acid change as compared to the sequence of the wild-type Fc domain comprising SEQ ID NO: 162. An amino acid change in an Fc region of an antibody construct can allow the antibody construct to bind to at least one Fc receptor with greater affinity compared to a wild-type Fc region. An amino acid change in an Fc domain of an antibody construct can allow the antibody to bind to at least one Fc receptor with greater affinity compared to a wild-type Fc domain. An Fc region can comprise an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to the natural or original amino acid sequence. An Fc domain can comprise an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine or ten modifications but not more than 40, 35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to the natural or original amino acid sequence. An Fc region can be an Fc region of an anti-CD40 antibody. An Fc domain can be an Fc domain of an anti-CD40 antibody. An Fc region can contain an Fc domain. An Fc region can be an Fc domain.
[0219] An antibody construct can be an antibody comprising a sequence of the IgGl isoform that has been modified from the wild type IgGl sequence. A wild type IgGl sequence can comprise SEQ ID NO: 162. A modification can comprise a substitution at more than one amino acid residue such as at 5 different amino acid residues including L235V/F243L/R292P/Y300L/P396L (G1VLPLL). The numbering of amino acids residues described herein can be according to the EU index as in Kabat. The 5 amino acid residues can be located in a portion of an antibody sequence which can encode an Fc region of the antibody and in particular, can be located in portions of the Fc region that can bind to Fc receptors (i.e., the Fc domain). A modification can comprise a substitution at more than one amino acid residue such as at 2 different amino acid residues including S239D/I332E (G1DE). The 2 amino acid residues can be located in a portion of an antibody sequence which encodes an Fc region of the antibody and in particular, are located in portions of the Fc region that can bind to Fc receptors (i.e., the Fc domain). A modification can comprise a substitution at more than one amino acid
-86residue such as at 3 different amino acid residues including S298A/E333A/K334A (G1 AAA). The 3 amino acid residues can be located in a portion of an antibody sequence which can encode an Fc region of the antibody and in particular, can be located in portions of the Fc region that can bind Fc receptors (i.e., the Fc domain).
[0220] An antibody construct can be a monoclonal anti-CD40 human antibody comprising a sequence of the IgGl isoform that has been modified from the wildtype IgGl sequence. A wildtype IgGl sequence can comprise SEQ ID NO: 15. A modification can comprise a substitution at more than one amino acid residue such as at 5 different amino acid residues including
L235V/F243L/R292P/Y300L/P396L (SBT-040-G1VLPLL). The numbering of amino acids residues described herein can be according to the EU index as in Kabat. The 5 amino acid residues can be located in a portion of an antibody sequence which can encode an Fc region of the antibody and in particular, can be located in portions of the Fc region that can bind to Fc receptors (i.e., the Fc domain). A modification can comprise a substitution at more than one amino acid residue such as at different amino acid residues including S239D/I332E (SBT-040-G1DE). The 2 amino acid residues can be located in a portion of an antibody sequence which encodes an Fc region of the antibody and in particular, are located in portions of the Fc region that can bind to Fc receptors (i.e., the Fc domain). A modification can comprise a substitution at more than one amino acid residue such as at 3 different amino acid residues including S298A/E333A/K334A (SBT-040-G1 AAA). The amino acid residues can be located in a portion of an antibody sequence which can encode an Fc region of the antibody and in particular, can be located in portions of the Fc region that can bind Fc receptors (i.e., the Fc domain).
[0221] Binding of Fc receptors to an Fc region can be affected by amino acid substitutions. For example, FIGURE 4C illustrates SBT-040-G1VLPLL, which is an antibody with an amino acid sequence (SEQ ID NO: 16) of a heavy chain of human anti-CD40 monoclonal antibody with modifications to a wild-type IgGl Fc domain (L235V/F243L/R292P/Y300L/P396L). Binding of some Fc receptors to the Fc region of SBT-040-G1VLPLL can be enhanced compared to wild-type by as result of the L235V/F243L/R292P/Y300L/P396L amino acid modifications. However, binding of other Fc receptors to the Fc region of SBT-040-G1VLPLL can be reduced compared to wild-type by the L235V/F243L/R292P/Y300L/P396L amino acid modifications. For example, the binding affinities of SBT-040-G1VLPLL to FcyRIIIA and to FcyRIIA can be enhanced compared to wildtype whereas the binding affinity of SBT-040-G1 VLPLLto FcyRIIB can be reduced compared to wild-type. FIGURE 4D illustrates an SBT-040-G1DE antibody, which is an antibody with an amino acid sequence (SEQ ID NO: 17) of a heavy chain of human anti-CD40 monoclonal antibody with
-87modifications to a wild-type IgGl Fc domain (S239D/I332E). Binding of Fc receptors to the Fc region of SBT-040-DE can be enhanced compared to wild-type as a result of the S239D/I332E amino acid modification. However, binding of some Fc receptors to the Fc region of SBT-040G1DE can be reduced compared to wild-type by S239D/I332E amino acid modification. For example, the binding affinities of SBT-040-G1DE to FcyRIIIA and to FcyRIIB can be enhanced compared to wild-type. Binding of Fc receptors to an Fc region of are affected by amino acid substitutions. FIGURE 4E illustrates an SBT-040-G1 AAA antibody, which is an antibody with an amino acid sequence (SEQ ID NO: 18) of a heavy chain of a human anti-CD40 monoclonal antibody with modifications to a wild-type IgGl Fc domain (S298A/E333A/K334A). Binding of Fc receptors to an Fc region of SBT-040-G1 AAA can be enhanced compared to wild-type as a result of the S298A/E333A/K334A amino acid modification. However, binding of some Fc receptors to the Fc region of SBT-040-G1 AAA can be reduced compared to wild-type by S298A/E333A/K334A amino acid modification. Binding affinities of SBT-040-G1 AAA to FcyRIIIA can be enhanced compared to wild-type whereas the binding affinity of SBT-040-G1 AAA to Fc/RIIB can be reduced compared to wildtype.
[0222] In some embodiments, the heavy chain of a human IgG2 antibody can be mutated at cysteines as positions 127, 232, or 233. In some embodiments, the light chain of a human IgG2 antibody can be mutated at a cysteine at position 214. The mutations in the heavy and light chains of the human IgG2 antibody can be from a cysteine residue to a serine residue.
[0223] An antibody construct can be a heavy chain of an anti-CD40 antibody. A heavy chain of an anti-CD40 antibody can be SBT-040-G1VFPFF. SBT-040-G1VFPFF be expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCA
GGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTC
TCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAAC
TATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAG
CCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
-88GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGC
CCACCGTGCCCAGCACCTGAACTCGTGGGGGGACCGTCAGTCTTCCTCCTGCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAAGCCGCCTGAGGAGCAGTACAACAGCACGCTGCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCTGGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCCCCGGGTAAATGA (SEQ ID NO: 9) wherein the DNA sequence comprises DNA nucleotide modifications that correspond to L235V, F243L, R292P, Y300L and P396L amino acid residue modifications compared to a wild-type DNA sequence. SBT-040-G1VLPLL can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 9. A variable region of SBT-040-G1VLPLL can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G1 VLPLL can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40 antibodies comprising SBT-040-G1VLPLL expressed from SEQ ID NO: 9, or expressed from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 9 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040G1 VLPLL expressed from DNA sequence comprising SEQ ID NO: 9, or comprising greater than 70% homology to SEQ ID NO: 9 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1 VLPLL can be expressed with any anti-CD40 light chain or fragment thereof. SBT-040-G1 VLPLL can also be expressed with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody
-89constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0224] SBT-040-G1VLPLL can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP LVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16) wherein the amino acid sequence comprises L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications compared to a wild-type amino acid sequence. SBT-040-G1VLPLL can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80?zo, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 16. SBT-040-G1 VLPLL can comprise an amino acid sequence SEQ ID NO: 20. A variable region of SBT-040-G1 VLPLL can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20. Additionally, anti-CD40 antibodies comprising SBT-040-G1 VLPLL with SEQ ID NO: 16 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 16 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1 VLPLL with SEQ ID NO: 16 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 16 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT040-G1VLPLL can be purified. SBT-040-G1 VLPLL can be combined with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising antiCD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0225] A heavy chain of an anti-CD40 antibody can be SBT-040-G1DE. SBT-040-G1DE be expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCA
-90GGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTC
TCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAAC
TATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAG
CCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGC
CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGGATGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGCCCTCCCAGCCCCCGAGGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCCCCGGGTAAATGA (SEQ ID NO: 10) wherein the DNA sequence comprises DNA nucleotide modifications that correspond to S239D and I332E amino acid residue modifications compared to a wild-type DNA sequence. SBT-040-G1DE can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 10. A variable region of SBT040-G1DE can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G1DE can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40 antibodies comprising SBT-040-G1DE expressed from SEQ ID NO: 10, or expressed from a DNA sequence comprising greater than 70%
-91homology to SEQ ID NO: 10 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1DE expressed from DNA sequence comprising SEQ ID NO: 10, or comprising greater than 70% homology to SEQ ID NO: 10 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1DE can be expressed with any anti-CD40 light chain or fragment thereof. SBT-040-G1DE can also be expressed with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0226] SBT-040-G1DE can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPE EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17) wherein the amino acid sequence comprises S239D and I332E amino acid residue modifications compared to a wild-type amino acid sequence. SBT-040-G1DE can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 17. SBT-040G1DE can comprise an amino acid sequence SEQ ID NO: 20. A variable region of SBT-040-G1DE can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO:
20. Additionally, anti-CD40 antibodies comprising SBT-040-G1DE with SEQ ID NO: 17 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 17 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1DE with SEQ ID NO: 17 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 17 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1DE can be purified. SBT-040-G1DE
-92can be combined with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0227] A heavy chain of an anti-CD40 antibody can be SBT-040-G1 AAA. SBT-040-G1 AAA be expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCCCA
GGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTC
TCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGC
CCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAAC
TATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAG
CCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGA
GATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG
CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGACAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGC
CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACG
TGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA
TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACGCCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGCCCTCCCAGCCCCCATCGCCGCTACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC
TCCCTGTCCCCGGGTAAATGA (SEQ ID NO: 11) wherein the DNA sequence comprises DNA
-93nucleotide modifications that correspond to S298A, E333A, and K334A amino acid residue modifications compared to a wild-type DNA sequence. SBT-040-G1 AAA can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 1E A variable region of SBT-040-G1 AAA can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G1 AAA can be expressed from a DNA sequence comprising greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40 antibodies comprising SBT-040-G1AAA expressed from SEQ ID NO: 11, or expressed from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 11 can have a dissociation constant (Ka) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1 AAA expressed from DNA sequence comprising SEQ ID NO: 11, or comprising greater than 70% homology to SEQ ID NO: 11 can have a dissociation constant (Ka) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1AAA can be expressed with any anti-CD40 light chain or fragment thereof. SBT-040-G1 AAA can also be expressed with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to antibody constructs comprising antiCD40 antibodies created for use in the veterinary sciences and/or in laboratory animals. SBT-040-G1AAA can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV I<FNWYVDGVEVHNAI<TI<PREEQYNATYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPI AATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) wherein the amino acid sequence comprises S298A, E333A, and K334A amino acid residue modifications compared to a wild-type amino acid sequence. SBT-040-G1 AAA can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 18. SBT-040-94G1 AAA can comprise an amino acid sequence SEQ ID NO: 20. A variable region of SBT-040G1 AAA can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20. Additionally, anti- CD40 antibodies comprising SBT-040-G1 AAA with SEQ ID NO: 18 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 18 can have a dissociation constant (Kd) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT040-G1AAA with SEQ ID NO: 18 or with an amino acid sequence with greater than 70% homology to SEQ ID NO: 18 can have a dissociation constant (Kd) for CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1AAA can be purified. SBT-040-G1 AAA can be combined with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can be purified, and can be combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody construct. Additionally, one skilled in the art would recognize that these same concepts could apply to anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
[0228] While an antibody construct of the present disclosure can comprise an anti-CD40 antibody with wild-type or modified amino acid sequences encoding the Fc region or Fc domain, the modifications of the Fc region or the Fc domain from the wild-type sequence may not significantly alter binding and/or affinity of the anti-CD40 antibody for CD40. For example, binding and/or affinity of SBT-040-G1WT, SBT-040-G1VLPLL, SBT-040-G1DE, and SBT-040-G1AAA may not be significantly altered by modification of an Fc region or Fc domain amino acid sequence compared to a wild-type sequence. Modifications of an Fc region or Fc domain from a wild-type sequence may not alter binding and/or affinity of antibodies that bind to CD40 in an antibody construct. Additionally, the binding and/or affinity of the antibodies described herein that bind to CD40 and are antibody constructs, for example SBT-040-G1WT, SBT-040-G1VLPLL, SBT-040-G1DE, and SBT040-G1 AAA, may be comparable to the binding and/or affinity of wild-type antibodies that can bind to CD40.
[0229] Sequences that can be used to produce antibodies for antibody constructs can include leader sequences. Leader sequences can be signal sequences. Leader sequences useful with the compositions and methods described herein can include, but are not limited to, a DNA sequence comprising
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGC (SEQ ID NO: 2) or
-95ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC (SEQ ID NO: 12), or an amino acid sequence comprising MRLPAQLLGLLLLWFPGSRC (SEQ ID NO: 5) and MDWTWRIFFFVAAATGAHS (SEQ ID NO: 19). Deader sequence can comprise a DNA sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 2 or SEQ ID NO: 12. Deader sequence can comprise an amino acid sequence with greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 5 or SEQ ID NO: 19. Any of the sequences described herein can be used with or without a leader sequence. Additionally, one skilled in the art would recognize that these same concepts can apply to antibody constructs comprising anti-CD40 antibodies created for use in the veterinary sciences and/or in laboratory animals.
Targeting Binding Domain [0230] An antibody construct can further comprise a targeting binding domain. A targeting domain can comprise a domain that binds to a target. A target can be an antigen. A targeting domain can comprise an antigen binding domain. A targeting domain can be a domain that can specifically bind to an antigen A targeting domain can be an antigen-binding portion of an antibody or an antibody fragment. A targeting domain can be one or more fragments of an antibody that can retain the ability to specifically bind to an antigen. A targeting domain can be any antigen binding fragment. A targeting domain can be in a scaffold, in which a scaffold is a supporting framework for the antigen binding domain. A targeting domain can comprise an antigen binding domain in a scaffold.
[0231] A targeting domain can comprise an antigen binding domain which can refer to a portion of an antibody comprising the antigen recognition portion, i.e., an antigenic determining variable region of an antibody sufficient to confer recognition and binding of the antigen recognition portion to a target, such as an antigen, i.e., the epitope. Examples of a targeting domain can include, but are not limited to, Fab, single chain variable fragment (scFv), variable Fv fragment and other fragments, combinations of fragments or types of fragments known or knowable to one of ordinary skill in the art. A targeting domain can comprise an antigen binding domain which can refer to a portion of an antibody comprising the antigen recognition portion, i.e., an antigenic determining variable region of an antibody sufficient to confer recognition and binding of the antigen recognition portion to a target, such as an antigen, i.e., the epitope. Examples of a targeting domain can include, but are not limited to, Fab, single chain variable fragment (scFv), variable Fv fragment and other fragments,
-96combinations of fragments or types of fragments known or knowable to one of ordinary skill in the art.
[0232] A targeting domain can comprise an antigen binding domain of an antibody. An antigen binding domain of an antibody can comprise one or more light chain (LC) CDRs and one or more heavy chain (HC) CDRs. For example, an antibody binding domain of an antibody can comprise one or more of the following: a light chain complementary determining region 1 (LC CDR1), a light chain complementary determining region 2 (LC CDR2), or a light chain complementary determining region 3 (LC CDR3). For another example, an antibody binding domain can comprise one or more of the following: a heavy chain complementary determining region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC CDR2), or a heavy chain complementary determining region 3 (HC CDR3).
[0233] An antibody construct can comprise an antibody fragment. An antibody fragment can include (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl and Chi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody. Although the two domains of the Fv fragment, Vl and Vh, can be coded for by separate genes, they can be linked by a synthetic linker to be made as a single protein chain in which the Vl and Vh regions pair to form monovalent molecules.
[0234] F(ab')2 and Fab' moieties can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and can include an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains.
[0235] An Fv can be the minimum antibody fragment which contains a complete antigenrecognition and antigen-binding site. This region can consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. In this configuration the three hypervariable regions of each variable domain can interact to define an antigen-binding site on the surface of the Vh-Vl dimer. A single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) can recognize and bind antigen, although at a lower affinity than the entire binding site.
[0236] A targeting domain can be at least 80% homologous to an antigen binding domain selected from, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or a functional fragment thereof, for example, a heavy chain variable domain (Vh) and a light chain variable domain (Vl), a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity clamp,
-97an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor, or a recombinant T cell receptor.
[0237] A targeting domain can comprise an antigen binding domain comprising a light chain and a heavy chain from a monoclonal antibody. In one aspect, a targeting domain binds to CD40 and comprises the light chain of an anti-CD40 antibody and the heavy chain of an anti-CD40 antibody, which bind a CD40 antigen. In another aspect, the targeting domain binds to a tumor antigen comprises the light chain of a tumor antigen antibody and the heavy chain of a tumor antigen antibody, which bind the tumor antigen.
[0238] A targeting domain can bet attached to an antibody construct. For example, an antibody construct can be fused with a targeting binding domain to create an antibody construct targeting binding domain fusion. The antibody construct- targeting binding domain fusion can be the result of the nucleic acid sequence of the targeting binding domain being expressed in frame with the nucleic acid sequence of the antibody construct. The antibody construct-targeting binding domain fusion can be the result of an in-frame genetic nucleotide sequence or a contiguous peptide sequence encoding the antibody construct with the targeting binding domain. As another example, a targeting binding domain can be linked to an antibody construct. A targeting binding domain can be linked to an antibody construct by a chemical conjugation. The targeting binding domain can direct the antibody construct to, for example, a particular cell or cell type. A targeting binding domain of an antibody construct can be selected in order to recognize an antigen. For example, an antigen can be expressed on an immune cell. An antigen can be a peptide or fragment thereof. An antigen can be expressed on an antigen-presenting cell. An antigen can be expressed on a dendritic cell, a macrophage, or a B cell. An antigen can be CD40 and a targeting binding domain can recognize a CD40 antigen. A targeting binding domain can be a CD40 agonist. A targeting domain can recognize CD40 on, for example, an antigen-presenting cell. As another example, an antigen can be a tumor antigen. The tumor antigen can be any tumor antigen described herein.
Immune-Stimulatory Compounds [0239] Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A PRR can be membrane bound. A PRR can be cytosolic. A PRR can be a toll-like receptor (TLR). A PRR can be RIG-I-like receptor.
A PRR can be a receptor kinase. A PRR can be a C-type lectin receptor. A PRR can be a NOD-like receptor. A PRR can be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
-98[0240] A PRR agonist can be pathogen-associated molecular pattern (PAMP) molecule. A PAMP molecule can be a toll-like receptor agonist. A PRR agonist can be a toll-like receptor agonist. A toll-like receptor agonist can be any molecule that acts as an agonist to at least one toll-like receptor. A toll-like receptor agonist can be bacterial lipoprotein. A toll-like receptor agonist can be bacterial peptidoglycans. A toll-like receptor agonist can be double stranded RNA. A toll-like receptor agonist can be lipopolysaccharides. A toll-like receptor agonist can be bacterial flagella. A toll-like receptor agonist can be single stranded RNA. A toll-like receptor can be CpG DNA. A toll-like receptor agonist can be imiquimod. A toll-like receptor agonist can be CL307. A toll-like receptor agonist can be S-27609. A toll-like receptor agonist can be resiquimod. A toll-like receptor agonist can be UCIV150. A toll-like receptor agonist can be gardiquimod. A toll-like receptor agonist can be motolimod. A toll-like receptor agonist can be VTX-1463. A toll-like receptor agonist can be GS9620. A toll-like receptor agonist can be GSK2245035. A toll-like receptor agonist can be TMX101. A toll-like receptor agonist can be TMX-201. A toll-like receptor agonist can be TMX-202. A toll-like receptor agonist can be isatoribine. A toll-like receptor agonist can be AZD8848. A toll-like receptor agonist can be MED 19197. A toll-like receptor agonist can be 3M-051. A toll-like receptor agonist can be 3M-852. A toll-like receptor agonist can be 3M-052. A toll-like receptor agonist can be 3M-854A. A toll-like receptor agonist can be S-34240. A toll-like receptor agonist can be CL663. A RIG-I agonist can be KIN1 148. A RIG-I agonist can be SB-9200. A RIG-I agonist can be KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, or KIN2000. A toll-like receptor agonist can be KU34B.
[0241] A PRR agonist can be a damage-associated molecular pattern (DAMP) molecule. A DAMP molecule can be an intracellular protein. A DAMP molecule can be a heat-shock protein. A DAMP molecule can be an HMGB1 protein. A DAMP molecule can be a protein derived from the extracellular matrix that is generated after tissue injury. A DAMP molecule can be a hyaluronan fragment. A DAMP molecule can be DNA. A DAMP molecule can be RNA. A DAMP molecule can be an SI00 molecule. A DAMP molecule can be nucleotides. A DAMP molecule can be an ATP. A DAMP molecule can be nucleosides. A DAMP molecule can be an adenosine. A DAMP molecule can be uric acid.
[0242] Additionally, stimulator of interferon genes (STING) can act as a cytosolic DNA sensor wherein cytosolic DNA and unique bacterial nucleic acids called cyclic dinucleotides are recognized by STING, and therefore STING agonists. Interferon Regulatory Factor (IRF) agonist can be KIN100. Non-limiting examples of STING agonists include:
Figure GB2552041A_D0082
, wherein in some embodiments, Xi=X2=0; X3=G; X4=G; X5=CO(CH2)i2CH3; X6=2 TEAH; in some embodiments, Xi=X2=S [RP,RP]; X3=G; X4=A; X5=H; X6=2 TEAH; in some embodiments, Xi=X2=S [RP,RP] ; X3=A; X4=A; Xs=H; X6=2 Na; in some embodiments, Xi=X2=S [RP,RP]; X3=A; X4=A; X5=H; X6=2 NH4; and in some embodiments, Xi=X2=O ; X3=G; X4=A; X5=H; X6=2 TEAH,
Figure GB2552041A_D0083
•A,
SA J'S
-100H / &'*·**<
/ X <Srcssss£ ‘ Si issss/
Y<V,Z /·<
,·ϊί·
Figure GB2552041A_D0084
, wherein
Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H, R2=allyl; Ri=methyl,
R2=H; Ri=H, R2=methyl; R^ethyl, R2=H; Ri=H, R2=ethyl; R^propyl, R2=H; Ri=H, R2=propyl;
Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H, R2=myristoyl; Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
Figure GB2552041A_D0085
Figure GB2552041A_D0086
>·· -N •’’v.jjX»’''' .
η ?>
/ v
Y'-wV/
s.X* * V,
Figure GB2552041A_D0087
/\Λ.
Figure GB2552041A_D0088
wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H, R2=allyl; Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl, R2=H; Ri=H, R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H, R2=myristoyl; Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
-101MM:·
Figure GB2552041A_D0089
MN·;
>
Figure GB2552041A_D0090
Figure GB2552041A_D0091
Χ,.ίί·γ'-/
Z.
<W^!>
❖ V.
Figure GB2552041A_D0092
'·., wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H, R2=allyl; Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl, R2=H; Ri=H, R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H, R2=myristoyl; Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
Figure GB2552041A_D0093
, wherein each X is independently 0 or S, and R3 and R4 are each independently H or an optionally substituted straight chain alkyl of from 1 to 18 carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from 1-9 carbons, an optionally substituted alkynyl of from 1-9 carbons, or an optionally substituted aryl, wherein substitution(s), when present, may be independently selected from the group consisting of Ci-6 alkyl straight or branched chain, benzyl, halogen, trihalomethyl, Ci-6 alkoxy, —NO2, —NH2, —OH, =0, —COOR' where R' is H or lower alkyl, —CH20H, and —C0NH2, wherein R3 and R4 are not both H,
-102ο
Figure GB2552041A_D0094
, wherein Χι=Χ2=Ο; Xi=X2=S; or Χχ=Ο and X2=S,
ΝΗ·?
Figure GB2552041A_D0095
-103-
Figure GB2552041A_D0096
Ν Η >>
-104-
Figure GB2552041A_D0097
<5
Ο
Figure GB2552041A_D0098
ο
Figure GB2552041A_D0099
[0243] An immune-stimulatory compound can be a PRR agonist. An immune-stimulatory compound can be a PAMP. An immune-stimulatory compound can be a DAMP. An immune
-105stimulatory compound can be a TLR agonist. An immune-stimulatory compound can be a STING agonist. An immune-stimulatory compound can be a cyclic dinucleotide.
[0244] An immune-stimulatory compound can be a drug.
[0245] The specificity of the antigen-binding domain to an antigen of an antibody construct in an antibody construct immune-stimulatory compound conjugate as disclosed herein can be influenced by the presence of an immune-stimulatory compound. The antigen-binding domain of the antibody construct in an antibody construct immune-stimulatory compound conjugate can bind to an antigen with at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of a specificity of the antigen-binding domain to the antigen in the absence of the immune-stimulatory compound.
[0246] The specificity of the Fc domain to an Fc receptor of an antibody construct in an antibody construct immune-stimulatory compound conjugate as disclosed herein can be influenced by the presence of an immune-stimulatory compound. The Fc domain of the antibody construct in an antibody construct immune-stimulatory compound conjugate can bind to an Fc receptor with at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of a specificity of the Fc domain to the Fc receptor in the absence of the immune-stimulatory compound.
[0247] The affinity of the antigen-binding domain to an antigen of an antibody construct in an antibody construct immune-stimulatory compound conjugate as disclosed herein can be influenced by the presence of an immune-stimulatory compound. The antigen-binding domain of the antibody construct in an antibody construct immune-stimulatory compound conjugate can bind to an antigen with at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of an affinity of the antigen-binding domain to the antigen in the absence of the immune-stimulatory compound.
[0248] The affinity of the Fc domain to an Fc receptor of an antibody construct in an antibody construct immune-stimulatory compound conjugate as disclosed herein can be influenced by the presence of an immune-stimulatory compound. The Fc domain of the antibody construct in an antibody construct immune-stimulatory compound conjugate can bind to an Fc receptor with at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of an affinity of the Fc domain to the Fc receptor in the absence of the immune-stimulatory compound.
[0249] The Ka for binding of an antigen-binding domain of an antibody construct immunestimulatory compound conjugate to an antigen in the presence of an immune-stimulatory compound
-106can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the Ka for binding of the antigen binding domain to the antigen of an antibody construct in the absence of the immune-stimulatory compound. The Ka for binding of an antigen-binding domain of an antibody construct immune-stimulatory compound conjugate to an antigen in the presence of the immune-stimulatory compound can be less than 10 nM. The Ka for binding of an antigen-binding domain of an antibody construct immune-stimulatory compound conjugate to an antigen in the presence of the immune-stimulatory compound can be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM.
[0250] The Ka for binding of an Fc domain of an antibody construct immune-stimulatory compound conjugate to a Fc receptor in the presence of the immune-stimulatory compound can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the Ka for binding of the Fc domain to the Fc receptor in the absence of the immune-stimulatory compound.
The Ka for binding of an Fc domain of an antibody construct immune-stimulatory compound conjugate to an Fc receptor in the presence of the immune-stimulatory compound can be less than 10 nM. The Ka for binding of an Fc domain of an antibody construct immune-stimulatory compound conjugate to an Fc receptor in the presence of the immune-stimulatory compound can be less than 10μ M, less than 1 μΜ, less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM.
[0251] Affinity can be the strength of the sum total of noncovalent interactions between a single binding site of a molecule, for example, an antibody, and the binding partner of the molecule, for example, an antigen. The affinity can also measure the strength of an interaction between an Fc portion of an antibody and the Fc receptor. Unless indicated otherwise, as used herein, “binding affinity” can refer to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or Fc domain and Fc receptor). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Ka). Affinity can be measured by common methods known in the art, including those described herein. Specific
-107illustrative and exemplary embodiments for measuring binding affinity are described in the following.
[0252] In some embodiments, an antibody provided herein can have a dissociation constant (Ka) of about 1 μΜ, about 100 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM, about 0.01 nM, or about 0.001 nM or less (e.g., 10'8 M or less, e.g., from 10' 8M to 10'13 M, e.g., from 10'9 M to 10'13 M). An affinity matured antibody can be an antibody with one or more alterations in one or more complementarity determining regions (CDRs), compared to a parent antibody, which may not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen. These antibodies can bind to their antigen with a Ka of about 5χ 10'9 M, about 2χ 10'9 M, about 1 χ 10'9 M, about 5χ 10'1 M, about 2χ 10'9 M, about 1χ10'1θ M, about 5χ10'π M, about ΙχΙΟ'11 M, about 5χ10'12 M, about ΙχΙΟ'12 M, or less. In some embodiments, the antibody construct can have an increased affinity of at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or greater as compared to an antibody construct without alterations in one or more complementarity determining regions.
[0253] Ka can be measured by any suitable assay. For example, Kd can be measured by a radiolabeled antigen binding assay (RIA). For example, Kd can be measured using surface plasmon resonance assays (e.g, using a BIACORE®-2000 or a BIACORE®-3000).
[0254] Agonism can be described as the binding of a chemical to a receptor to induce a biological response. A chemical can be, for example, a small molecule, a compound, or a protein. An agonist causes a response, an antagonist can block the action of an agonist, and an inverse agonist can cause a response that is opposite to that of the agonist. A receptor can be activated by either endogenous or exogenous agonists.
[0255] The molar ratio of an antibody construct immune-stimulatory compound conjugate can refer to the average number of immune-stimulatory compounds conjugated to the antibody construct in a preparation of an antibody construct immune-stimulatory compound conjugate. The molar ratio can be determined, for example, by Liquid Chromatography/Mass Spectrometry (LC/MS), in which the number of immune-stimulatory compounds conjugated to the antibody construct can be directly determined. Additionally, as non-limiting examples, the molar ratio can be determined based on hydrophobic interaction chromatography (HIC) peak area, by liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS), by UV/Vis spectroscopy, by reversedphase-HPLC (RP-HPLC), or by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).
-108[0256] In some embodiments, the molar ratio of immune-stimulatory compound to antibody can be less than 8. In other embodiments, the molar ratio of immune-stimulatory compound to antibody can be 8, 7, 6, 5,4,3,2, or 1.
[0257] In some aspects, the present disclosure provides a compound represented by the structure of Formula (I):
Figure GB2552041A_D0100
or a pharmaceutically acceptable salt thereof, wherein:
X1 is selected from -OR2 and -SR2;
X2 is selected from -OR3 and -SR3;
B and B are independently selected from optionally substituted nitrogenous bases;
Y is selected from —OR4, -NR4R4, and halogen;
R1, R2, R3 and R4 are independently selected at each occurrence from hydrogen, -C(=O)R100,
C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2-io alkenyl, C2-10 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R , R , R and R is independently optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Cm alkyl, C2.6 alkenyl, and C2.6 alkynyl; and R100 at each occurrence is independently selected from hydrogen; and Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO2, =0, =S, and haloalkyl.
[0258] In some embodiments, the compound of Formula (I) is represented by Formula (IA):
-109-
Figure GB2552041A_D0101
or pharmaceutically acceptable salts thereof.
[0259] In an alternative embodiment, the compound of Formula (I) is represented by Formula (IB):
Figure GB2552041A_D0102
or a pharmaceutically acceptable salt thereof.
2 [0260] In various embodiments, B and B are independently selected from optionally substituted
2 purines. In certain embodiments, B and B are independently selected from: H . In certain embodiments, B and B are independently selected from optionally substituted pyrimidines.
[0261] In some embodiments, optionally substituted purines may include optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxaanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, and optionally substituted isoguanine. In certain embodiments, B and B are independently selected from:
-110Ν'
Η
Figure GB2552041A_D0103
ηνΑΛ
Η
Ν'‘'Τι ΝΗ °ΛΑα
ΝΗ
Ν , and Η νη2
ΝΗ
Ν A
C0:
Ν CH3
AM h/>
Ν CH3 , optionally substituted by one or more additional substituents.
NH2 na<^n [0262] In certain embodiments, B1 and B2 are independently selected from: >~L <'U
A o
N^N^NH2
JVVV nh2 'NH 'nA nm r, a /, Am „ /NT ΝΗ ?Ά^Νπ aA? YU °va ίAao , wherein the point of connectivity of B1 to the remainder of the compound is represented by .
2 [0263] In a preferred embodiment, B and B are independently selected from optionally substituted
2 adenine and optionally substituted guanine. In certain embodiments, B and B are independently NH2 O
N^n^-nh,
N selected from: H and H optionally further substituted by one or more NH2 nA
N substituents. In certain embodiments, B1 and B2 are independently selected from: Ά, O
Ah . .A.
N NH2
ΙΝΓ
A· and
2 [0264] In some embodiments, B and B are independently optionally substituted with one or more
2 substituents, wherein the optional substituents on B and B are independently selected at each occurrence from halogen, =0, =S, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, -P(O)(OR100)2, -OP(O)(OR100)2 and -CN; Cmo alkyl, C2.
-Illio alkenyl, C2-10 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle is independently optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN,
Ci_6 alkyl, C2-6 alkenyl, and C2-6 alkynyl.
2 [0265] In certain embodiments, B and B are independently optionally substituted with one or more
2 substituents, wherein the optional substituents on B and B are independently selected at each occurrence from halogen, =0, =S, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, -P(O)(OR100)2, -OP(O)(OR100)2, -CN and Cmo alkyl. [0266] In some embodiments, B1 is an optionally substituted guanine. In certain embodiments, B1 is
O . In certain embodiments, B
O , wherein the point of connectivity of B1 to the remainder of the compound is represented by X . In some embodiments, B1 is an NH2 optionally substituted adenine. In certain embodiments, B1 is NH2 . In certain embodiments, wherein the point of connectivity of B1 to the remainder of the compound is represented by utjLv
2 [0267] In some embodiments, B is an optionally substituted guanine. In certain, embodiments, B is
O . In certain embodiments, B
O , wherein the point of connectivity on B2 is represented by X . In some embodiments, B2 is an optionally substituted adenine. In certain
-112. In certain embodiments, B2 is
N embodiments, B2 is H nh2
N
N
C0 nh2
N
N
C0 wherein the point of connectivity on B is represented by
2 [0268] In some embodiments, B is an optionally substituted guanine and B is an optionally substituted guanine. In some embodiments, B is an optionally substituted adenine and B is an optionally substituted guanine.
[0269] In various embodiments, X1 is selected from -OH and -SH. For example, X1 may be -OH. In various embodiments, X is selected from -OH and -SH. For example, X may be -OH. In some embodiments, X is -OH and X is -OH. In some embodiments, X is -SH and X is -SH.
[0270] In various embodiments, 5/ is selected from OH, O-Ci-iq alkyl, F1H(Ci-iq alkyl), and hiFF. For example, Y may be -OH.
[0271] In various embodiments, R100 is independently selected at each occurrence from hydrogen and Cmo alkyl optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO2, =0, and =S.
[0272] In various embodiments, the compound of Formula (I) is represented by Formula (IC):
Figure GB2552041A_D0104
or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (IC) is represented by Formula (ID):
Figure GB2552041A_D0105
or a pharmaceutically acceptable salt thereof.
-113[0273] In various embodiments, the compound is a pharmaceutically acceptable salt. In some embodiments, the compound or salt is a modulator of a stimulator of interferon genes (STING). The compound or salt may agonize a stimulator of interferon genes (STING). In certain embodiments, the compound or salt may cause STING to coordinate multiple immune responses to infection, including the induction of interferons and STAT6-dependent response and selective autophagy response. In certain embodiments, the compound or salt may cause STING to mediate type I interferon production.
[0274] In some aspects, the present disclosure provides an antibody drug conjugate, comprising a compound or salt previously described, an antibody, and a linker group, wherein the compound or salt is linked, e.g., covalently bound, to the antibody through the linker group. The linker group may be selected from a cleavable or non-cleavable linker. In some embodiments, the linker group is cleavable. In alternative embodiments, the linker group is non-cleavable. Linkers are further described in the present application in the subsequent section, any one of which may be used to connect an antibody to a compound described herein.
[0275] In some aspects, the present disclosure provides a compound represented by the structure of Formula (II):
B·
Figure GB2552041A_D0106
Figure GB2552041A_D0107
or a pharmaceutically acceptable salt thereof, wherein:
X1 is selected from -OR2 and -SR2;
X2 is selected from -OR3 and -SR3;
2
B and B are independently selected from optionally substituted nitrogenous bases, wherein each optional substituent is independently selected from halogen, -OR100, -SR100, -N(R100)2, -
Figure GB2552041A_D0108
Y is selected from —OR4, -SR4, -NR4R4, and halogen; Z is selected from —OR5, -SR5, and -NR5R5;
-114R1, R2, R3, R4, and R5 are independently selected from a -X3; hydrogen, -C(=O)R100, C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R , R , R , R , and R is optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100-C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci^ alkyl, C2.6 alkenyl, C2.6 alkynyl;
R6 is independently selected from -C(=O)R100, -C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R6 is optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci^ alkyl, C2.6 alkenyl, C2-6 alkynyl;
R100 at each occurrence is independently selected from hydrogen; and Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO2, =0, =S, and haloalkyl; and
X is a linker moiety, wherein at least one ofR,R,R,R,R,X,X,aB substituent and a
3
B substituent is -X .
[0276] In various embodiments, the compound of Formula (II) is represented by a structure of Formula (IIA):
-115-
Figure GB2552041A_D0109
(HA) ?
or pharmaceutically acceptable salts thereof.
[0277] In various embodiments, the compound of Formula (II) is represented by a structure of Formula (IIB):
Figure GB2552041A_D0110
or a pharmaceutically acceptable salt thereof.
[0278] In various embodiments, B and B are independently selected from optionally substituted purines. B1 and B2 may be each, independently selected from one another, adenine, guanine, and derivatives thereof. B and B may be independently selected from optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, and optionally substituted isoguanine. In a preferred embodiment, B and B are independently selected from optionally substituted adenine and optionally substituted guanine.
[0279] In various embodiments, B is substituted by X and optionally one or more additional substituents independently selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, and R6. For example,
-116HN <;« y N 1
B may be represented by: , and wherein B is optionally further substituted by one or more substituents.
3 [0280] In various embodiments, B is substituted by X and optionally one or more additional substituents independently selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, and R6. For example,
B2 may be represented by: more substituents.
HN
Ν N 'y , and wherein B is optionally further substituted by one or
HN
Ν N [0281] In some embodiments, B1 is represented by V/v /X3
HN
Ν N
NH2 <;ό
7 N and B is represented by o
NH
N' NH2
In some embodiments, B1 is represented by and B2 is represented by 5,
3 3 1 [0282] In various embodiments, X is selected from -Ο- X and-S-X . In some embodiments, X is selected from -OH and -SH. In some embodiments, X1 is -SH.
3 3 2 [0283] In various embodiments, X is selected from -Ο- X and-S-X . In some embodiments, X is
3 selected from -OH and -SH. In some embodiments, X is -S-X .
[0284] In some embodiments, X1 is -SH and X2 is -S-X3.
3 3 3 [0285] In certain embodiments, Y is selected from -NR X , -S-X , and -Ο- X . In some embodiments, Y is selected from -OH, -SH, -O-Cmo alkyl, -NH(Cmo alkyl), and -NH2. In a preferred embodiment, Y is selected from -OH.
3 3 3 [0286] In various embodiments, Z is selected from -NR X , -S-X , and -Ο- X . In some embodiments, Z is selected from -OH, -SH, -O-Cmo alkyl, -NH(Cmo alkyl), and -NH2
-117β [0287] In various embodiments, -X is represented by the formula:
o
A.
Figure GB2552041A_D0111
O
A
N peptide H
Figure GB2552041A_D0112
O
Λ β
[0288] In some embodiments, -X is represented by the formula: , wherein RX comprises a reactive moiety, such a maleimide.
β [0289] In some embodiments, -X is represented by the formula:
peptide-RX o
A.
Figure GB2552041A_D0113
o
A
N peptide-RX*-Antibody
H ', wherein RX is a reactive moiety that has reacted with a moiety on an antibody to form an antibody-drug conjugate.
β [0290] In some embodiments, -X is represented by the formula:
Figure GB2552041A_D0114
RX , wherein RX is a reactive moiety, such as a maleimide.
β [0291] In some embodiments, -X is represented by the formula:
Figure GB2552041A_D0115
z RX*-Antibody * /1_4 H , wherein RX is a reactive moiety that has reacted with a moiety on an antibody to form an antibody drug conjugate.
[0292] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0116
o
-118or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0117
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula: o
Figure GB2552041A_D0118
O , or a pharmaceutically acceptable salt thereof.
[0293] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0119
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0120
CHs
Figure GB2552041A_D0121
, or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
-119ο
Figure GB2552041A_D0122
Figure GB2552041A_D0123
, or a pharmaceutically acceptable salt thereof.
[0294] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0124
thereof. The compound may be represented by the formula:
Figure GB2552041A_D0125
, or a pharmaceutically acceptable salt , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0126
0 , or a pharmaceutically acceptable salt thereof.
[0295] In some embodiments, the compound is represented by the formula:
o
Figure GB2552041A_D0127
o , or a pharmaceutically acceptable salt
-120thereof. The compound may be represented by the formula:
Figure GB2552041A_D0128
, or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0129
o , or a pharmaceutically acceptable salt thereof.
[0296] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0130
, or a pharmaceutically acceptable salt thereof.
[0297] The compound is represented by the formula:
Figure GB2552041A_D0131
o , or a pharmaceutically acceptable salt thereof.
-121[0298] The compound may be represented by the formula:
Figure GB2552041A_D0132
o , or a pharmaceutically acceptable salt thereof.
[0299] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0133
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0134
, or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0135
acceptable salt thereof.
, or a pharmaceutically
-122[0300] In some embodiments, the compound is represented by the formula:
x1p °hp h2n_n fj, °\_/
Figure GB2552041A_D0136
HNA 'Px„ ° , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
o
Figure GB2552041A_D0137
or a pharmaceutically actable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0138
Figure GB2552041A_D0139
o , or a pharmaceutically acceptable salt thereof.
[0301] In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0140
° , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
-123-
Figure GB2552041A_D0141
ο , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
Figure GB2552041A_D0142
° , or a pharmaceutically acceptable salt thereof.
In some embodiments, the compound is represented by the formula:
Figure GB2552041A_D0143
thereof. The compound may be represented by the formula:
, or a pharmaceutically acceptable salt
Figure GB2552041A_D0144
o , or a pharmaceutically acceptable salt thereof. The compound may be represented by the formula:
-124-
Figure GB2552041A_D0145
Ο , or a pharmaceutically acceptable salt thereof.
Linkers [0302] The compositions and methods described herein can comprise a linker, e.g., a peptide linker. Linkers of the compositions and methods described herein may not affect the binding of active portions of a conjugate (e.g, active portions include antigen binding domains, Fc domains, targeting binding domains, antibodies, agonists or the like) to a target, which can be a cognate binding partner such as an antigen. A linker sequence can form a linkage between different parts of a composition. A composition can be a conjugate. A conjugate can comprise multiple linkers. These linkers can be the same linkers or different linkers.
[0303] Attachment via a linker can involve incorporation of a linker between parts of a composition or conjugate. A linker can be short, flexible, rigid, cleavable, non-cleavable, hydrophilic, or hydrophobic. A linker can contain segments that have different characteristics, such as segments of flexibility or segments of rigidity. The linker can be chemically stable to extracellular environments, for example, chemically stable in the blood stream, or may include linkages that are not stable. The linker can include linkages that are designed to cleave and/or immolate or otherwise breakdown specifically or non- specifically inside cells. A cleavable linker can be sensitive to enzymes. A cleavable linker can be cleaved by enzymes such as proteases. A cleavable linker can be a valinecitrulline linker or a valine-alanine linker. A valine-citrulline or valine-alanine linker can contain a pentafluorophenyl group. A valine-citrulline or valine-alanine linker can contain a succimide group. A valine-citrulline or valine-alanine linker can contain a para aminobenzoic acid (PABA) group. A valine-citrulline or valine-alanine linker can contain a PABA group and a pentafluorophenyl group. A valine-citrulline or valine-alanine linker can contain a PABA group and a succinimide group. A non-cleavable linker can be protease insensitive. A non-cleavable linker can be maleimidocaproyl linker. A maleimidocaproyl linker can comprise N-maleimidomethylcyclohexane-1 -carboxylate. A maleimidocaproyl linker can contain a succinimide group. A maleimidocaproyl linker can contain pentafluorophenyl group. A linker can be a combination of a maleimidocaproyl group and one or
-125more polyethylene glycol molecules. A linker can be a maleimide-PEG4 linker. A linker can be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. A linker can be a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules. A linker can contain maleimides linked to polyethylene glycol molecules in which the polyethylene glycol can allow for more linker flexibility or can be used lengthen the linker. A linker can be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonly)-(NH2) linker. A linker can be a THIOMAB linker. A THIOMAB linker can be a (maleimidocaproyl)-(valine-citrulline)-(paraaminobenzyloxycarbonly)-(NH2) linker. A linker can also be an alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides, or aminobenzylcarbamates. A linker can contain a maleimide at one end and an Nhydroxysuccinimidyl ester at the other end. A linker can contain a lysine with an N-terminal amine acetylated, and a valine-citrulline cleavage site. A linker can be a link created by a microbial transglutaminase, wherein the link can be created between an amine-containing moiety and a moiety engineered to contain glutamine as a result of the enzyme catalyzing a bond formation between the acyl group of a glutamine side chain and the primary amine of a lysine chain. A linker can contain a reactive primary amine. A linker can be a Sortase A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG recognition motif (SEQ ID NO: 21) to an N-terminal GGG motif to regenerate a native amide bond. The linker created can therefore link a moiety attached to the LXPTG recognition motif (SEQ ID NO: 21) with a moiety attached to the N-terminal GGG motif. A linker can be a link created between an unnatural amino acid on one moiety reacting with oxime bond that was formed by modifying a ketone group with an alkoxyamine on another moiety. A moiety can be an antibody construct. A moiety can be an antibody. A moiety can be an immunestimulatory compound. A moiety can be a targeting binding domain. A linker can be a portion of a linker can be unsubstituted or substituted, for example, with a substituent. A substituent can include, for example, hydroxyl groups, amino groups, nitro groups, cyano groups, azido groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, acyl groups, acyloxy groups, amide groups, and ester groups.
[0304] In the antibody construct immune-stimulatory compound conjugate described herein, the immune-stimulatory compound is linked to the antibody construct by way of linkers. The linker linking an immune-stimulatory compound to the antibody of an antibody construct immunestimulatory compound conjugate can be short, long, hydrophobic, hydrophilic, flexible or rigid, or may be composed of segments that each independently have one or more of the above-mentioned
-126properties such that the linker may include segments having different properties. The linkers can be polyvalent such that they covalently link more than one immune-stimulatory compound to a single site on the antibody construct, or monovalent such that covalently they link a single immunestimulatory compound to a single site on the antibody construct.
[0305] As will be appreciated by skilled artisans, the linkers link the immune-stimulatory compound to the antibody by forming a covalent linkage to the immune-stimulatory compound at one location and a covalent linkage to the antibody construct at another. The covalent linkages are formed by reaction between functional groups on the linker and functional groups on the inhibitors and antibody construct. As used herein, the expression linker is intended to include (i) unconjugated forms of the linker that include a functional group capable of covalently linking the linker to an immune-stimulatory compound and a functional group capable of covalently linking the linker to an antibody construct; (ii) partially conjugated forms of the linker that include a functional group capable of covalently linking the linker to an antibody construct and that is covalently linked to an immune-stimulatory compound, or vice versa; and (iii) fully conjugated forms of the linker that is covalently linked to both an immune-stimulatory compound and an antibody construct. In some specific embodiments of intermediate synthons and antibody construct immune-stimulatory compound conjugates described herein, moieties comprising the functional groups on the linker and covalent linkages formed between the linker and antibody construct are specifically illustrated as Rx and LK, respectively. One embodiment pertains to an antibody construct immune-stimulatory compound conjugate formed by contacting an antibody construct that binds a cell surface receptor or tumor associated antigen expressed on a tumor cell with a synthon described herein under conditions in which the synthon covalently links to the antibody construct. One embodiment pertains to a method of making an antibody construct immune-stimulatory compound conjugate formed by contacting a synthon described herein under conditions in which the synthon covalently links to the antibody construct. One embodiment pertains to a method of stimulating immune activity in a cell that expresses CD40, comprising contacting the cell with an antibody construct immune-stimulatory compound conjugate described herein that is capable of binding the cell, under conditions in which the antibody construct immune-stimulatory compound conjugate binds the cell.
[0306] Exemplary polyvalent linkers that may be used to link many immune-stimulatory compounds to an antibody construct are described. For example, Fleximer® linker technology has the potential to enable high-DAR antibody construct immune-stimulatory compound conjugate with good physicochemical properties. As shown below, the Fleximer® linker technology is based on incorporating drug molecules into a solubilizing poly-acetal backbone via a sequence of ester bonds.
-127The methodology renders highly-loaded antibody construct immune-stimulatory compound conjugates (DARup to 20) whilst maintaining good physicochemical properties. This methodology could be utilized with immune-stimulatory compound as shown in the Scheme below.
Figure GB2552041A_D0146
[0307] To utilize the Fleximer® linker technology depicted in the scheme above, an aliphatic alcohol can be present or introduced into the immune-stimulatory compound. The alcohol moiety is then conjugated to an alanine moiety, which is then synthetically incorporated into the Fleximer® linker. Liposomal processing of the antibody construct immune-stimulatory compound conjugate in vitro releases the parent alcohol-containing drug.
[0308] By way of example and not limitation, some cleavable and noncleavable linkers that may be included in the antibody construct immune-stimulatory compound conjugates described herein are described below.
[0309] Cleavable linkers can be cleavable in vitro and in vivo. Cleavable linkers can include chemically or enzymatically unstable or degradable linkages. Cleavable linkers can rely on processes inside the cell to liberate an immune-stimulatory compound, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes
-128within the cell. Cleavable linkers can incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker can be non-cleavable.
[0310] A linker can contain a chemically labile group such as hydrazone and/or disulfide groups. Linkers comprising chemically labile groups can exploit differential properties between the plasma and some cytoplasmic compartments. The intracellular conditions that can facilitate immunestimulatory compound release for hydrazone containing linkers can be the acidic environment of endosomes and lysosomes, while the disulfide containing linkers can be reduced in the cytosol, which can contain high thiol concentrations, e.g., glutathione. The plasma stability of a linker containing a chemically labile group can be increased by introducing steric hindrance using substituents near the chemically labile group.
[0311] Acid-labile groups, such as hydrazone, can remain intact during systemic circulation in the blood’s neutral pH environment (pH 7.3-7.5) and can undergo hydrolysis and can release the immune-stimulatory compound once the antibody construct immune-stimulatory compound conjugate is internalized into mildly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of the cell. This pH dependent release mechanism can be associated with nonspecific release of the drug. To increase the stability of the hydrazone group of the linker, the linker can be varied by chemical modification, e.g., substitution, allowing tuning to achieve more efficient release in the lysosome with a minimized loss in circulation.
[0312] Hydrazone-containing linkers can contain additional cleavage sites, such as additional acidlabile cleavage sites and/or enzymatically labile cleavage sites. Antibody construct immunestimulatory compound conjugates including exemplary hydrazone-containing linkers can include, for example, the following structures:
-129Ah (ig)
D'
JI
Jn
Figure GB2552041A_D0147
wherein D and Ab represent the immune-stimulatory compound and antibody construct, respectively, and n represents the number of immune-stimulatory compound - linkers linked to the antibody construct. In certain linkers such as linker (Ig), the linker can comprise two cleavable groups- a disulfide and a hydrazone moiety. For such linkers, effective release of the unmodified free immunestimulatory compound can require acidic pH or disulfide reduction and acidic pH. Linkers such as (Ih) and (Ii) can be effective with a single hydrazone cleavage site.
[0313] Other acid-labile groups that can be included in linkers include cA-aconityl-containing linkers. c/.s-Aconityl chemistry can use a carboxylic acid juxtaposed to an amide bond to accelerate amide hydrolysis under acidic conditions.
[0314] Cleavable linkers can also include a disulfide group. Disulfides can be thermodynamically stable at physiological pH and can be designed to release the immune-stimulatory compound upon internalization inside cells, wherein the cytosol can provide a significantly more reducing environment compared to the extracellular environment. Scission of disulfide bonds can require the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), such that disulfidecontaining linkers can be reasonably stable in circulation, selectively releasing the immune-130stimulatory compound in the cytosol. The intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, can also contribute to the preferential cleavage of disulfide bonds inside cells. GSH can be present in cells in the concentration range of 0.5-10 mM compared with a significantly lower concentration of GSH or cysteine, the most abundant lowmolecular weight thiol, in circulation at approximately 5 μΜ. Tumor cells, where irregular blood flow can lead to a hypoxic state, can result in enhanced activity of reductive enzymes and therefore even higher glutathione concentrations. The in vivo stability of a disulfide-containing linker can be enhanced by chemical modification of the linker, e.g., use of steric hindrance adjacent to the disulfide bond.
[0315] Antibody construct immune-stimulatory compound conjugates including exemplary disulfide-containing linkers can include the following structures:
R R
Figure GB2552041A_D0148
.S’
Ab
ET
:.O wherein D and Ab represent the immune-stimulatory compound and antibody construct, respectively, n represents the number of immune-stimulatory compound -linkers linked to the antibody construct and R is independently selected at each occurrence from hydrogen or alkyl, for example. Increasing steric hindrance adjacent to the disulfide bond can increase the stability of the linker. Structures such as (Ij) and (II) can show increased in vivo stability when one or more R groups is selected from a lower alkyl such as methyl.
-131[0316] Another type of linker that can be used is a linker that is specifically cleaved by an enzyme. For example, the linker can be cleaved by a lysosomal enzyme. Such linkers can be peptide-based or can include peptidic regions that can act as substrates for enzymes. Peptide based linkers can be more stable in plasma and extracellular milieu than chemically labile linkers.
[0317] Peptide bonds can have good serum stability, as lysosomal proteolytic enzymes can have very low activity in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes. Release of an immune-stimulatory compound from an antibody construct can occur due to the action of lysosomal proteases, e.g., cathepsin and plasmin. These proteases can be present at elevated levels in certain tumor tissues. The linker can be cleavable by a lysosomal enzyme. The lysosomal enzyme can be, for example, cathepsin B, β-glucuronidase, or βgalactosidase.
[0318] The cleavable peptide can be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-LeuAla-Leu or dipeptides such as Val-Cit, Val-Ala, and Phe-Lys. Dipeptides can have lower hydrophobicity compared to longer peptides.
[0319] A variety of dipeptide-based cleavable linkers can be used in the antibody constructs immune-stimulatory compound conjugates described herein.
[0320] Enzymatically cleavable linkers can include a self-immolative spacer to spatially separate the immune-stimulatory compound from the site of enzymatic cleavage. The direct attachment of an immune-stimulatory compound to a peptide linker can result in proteolytic release of an amino acid adduct of the immune-stimulatory compound, thereby impairing its activity. The use of a selfimmolative spacer can allow for the elimination of the fully active, chemically unmodified immunestimulatory compound upon amide bond hydrolysis.
[0321] One self-immolative spacer can be a bifunctional pr/ra-aminobenzyl alcohol group, which can link to the peptide through the amino group, forming an amide bond, while amine containing immune-stimulatory compounds can be attached through carbamate functionalities to the benzylic hydroxyl group of the linker (to give a /i-amidobenzylcarbamate, PABC). The resulting proimmune-stimulatory compound can be activated upon protease- mediated cleavage, leading to a 1,6elimination reaction releasing the unmodified immune-stimulatory compound, carbon dioxide, and remnants of the linker group. The following scheme depicts the fragmentation of p- amidobenzyl carbamate and release of the immune-stimulatory compound:
-132-
Figure GB2552041A_D0149
J X-D wherein X-D represents the unmodified immune-stimulatory compound.
Heterocyclic variants of this self-immolative group have also been described.
[0322] The enzymatically cleavable linker can be a B-glucuronic acid-based linker. Facile release of the immune-stimulatory compound can be realized through cleavage of the B-glucuronide glycosidic bond by the lysosomal enzyme B-glucuronidase. This enzyme can be abundantly present within lysosomes and can be overexpressed in some tumor types, while the enzyme activity outside cells can be low. B- Glucuronic acid-based linkers can be used to circumvent the tendency of an antibody construct immune-stimulatory compound conjugate to undergo aggregation due to the hydrophilic nature of B-glucuronides. In certain embodiments, B-glucuronic acid-based linkers can link an antibody construct to a hydrophobic immune-stimulatory compound. The following scheme depicts the release of an immune-stimulatory compound (D) from an antibody construct (Ab) immunestimulatory compound conjugate containing a B-glucuronic acid-based linker:
Figure GB2552041A_D0150
HO '·· jp^ M2
Ab
Figure GB2552041A_D0151
•••CO·· [0323] A variety of cleavable β-glucuronic acid-based linkers useful for linking drugs such as auristatins, camptothecin and doxorubicin analogues, CBI minor-groove binders, and psymberin to antibodies have been described. All of these β-glucuronic acid-based linkers may be used in the ADCs described herein. In certain embodiments, the enzymatically cleavable linker is a βgalactoside-based linker. β-Galactoside is present abundantly within lysosomes, while the enzyme activity outside cells is low.
[0324] Additionally, immune-stimulatory compounds containing a phenol group can be covalently bonded to a linker through the phenolic oxygen. One such linker relies on a methodology in which a
-133diamino-ethane Space Link is used in conjunction with traditional PABO -based self-immolative groups to deliver phenols.
[0325] Cleavable linkers can include non-cleavable portions or segments, and/or cleavable segments or portions can be included in an otherwise non-cleavable linker to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers can include cleavable groups in the polymer backbone. For example, a polyethylene glycol or polymer linker can include one or more cleavable groups such as a disulfide, a hydrazone or a dipeptide.
[0326] Other degradable linkages that can be included in linkers can include ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on an immune-stimulatory compound, wherein such ester groups can hydrolyze under physiological conditions to release the immune-stimulatory compound. Hydrolytically degradable linkages can include, but are not limited to, carbonate linkages; imine linkages resulting from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
[0327] A linker can contain an enzymatically cleavable peptide moiety, for example, a linker comprising structural formula (IVa), (IVb), (IVc), or (IVd):
-134(IVb)
Ra (IVc) (IW)
Ra
Ra I tM
Figure GB2552041A_D0152
-peptide—N H o
••χ. ..-Ok
Figure GB2552041A_D0153
X.
Rs 'peptide—N'
H
R* ^pept ide—N H
R?
J.
Ζ'-χ-f b-oA
Χ'·-ν .--^χ·*' % o A ck'U or a salt thereof, wherein: peptide represents a peptide (illustrated N—>C, wherein peptide includes the amino and carboxy “termini”) a cleavable by a lysosomal enzyme; T represents a polymer comprising one or more ethylene glycol units or an alkylene chain, or combinations thereof; Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; Ry is hydrogen or Cw alkyl-(O)r-(Ci. 4 alkylenejs-G1 or Cw alkyl-(N)-[(Ci-4 alkylenej-GX; Rz is Cw alkyl-(0)r-(Ci-4 alkylene)s-G2; G1 is SO3H, C02H, PEG 4-32, or sugar moiety; G2 is SO3H, C02H, or PEG 4-32 moiety; r is 0 or 1; s is 0 /
or 1; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1; $ represents the point of attachment of the linker to the immune-stimulatory compound; and * represents the point of attachment to the remainder of the linker.
[0328] In certain embodiments, the peptide can be selected from a tripeptide or a dipeptide. In particular embodiments, the dipeptide can be selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-135Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; CitAsp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu- Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit, or salts thereof.
[0329] Exemplary embodiments of linkers according to structural formula (IVa) that can be included in the antibody construct immune-stimulatory compound conjugates described herein can include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):
(IVa. I) (IVa J % i h h
Lit . fk. .-N.
'V V K* (IVaJ)
Figure GB2552041A_D0154
UVa.,4)
-136(i\W>
7a.7) ci
Figure GB2552041A_D0155
Figure GB2552041A_D0156
o
Figure GB2552041A_D0157
(IVaM « v,
S·, o , Q Va f SI h ll J n
I δ η 1 Μ η 1 [0330] Exemplary embodiments of linkers according to structural formula (IVb), (IVc), or (IVd) that can be included in the antibody construct immune-stimulatory compound conjugates described herein can include the linkers illustrated below (as illustrated, the linkers can include a group suitable for covalently linking the linker to an antibody construct):
-137uvb.n •α I K 9
An 'nA«*M *' ' h 1 ;H __X
x.
?·:>··
Ov NHs q V □ A%Afr
Α^α,Α,, q
JW
b.2)
RS-i κ>Λ
4.3) τΝ(β.χ*'*ΛΐΝν.^ί*'
Υ.κ.
o
Ά>:
--i.i
4)
Νχ ,/ xiSS^ q 2 Y K § r I :j-S X
A* □' fivb.s)
N:R.
<A»® 4
». i, 1 ' * Γ H ,Κ.^-'Χ,,·'·',. ·-Ά-'f ' *: -'Ά·-·''
V : !! £ :ri '>3...-/N-.
ANA ΝΑ Y
NH?
-138(IVb.fo ο
Λ* ϊ H :( Γ [ •v. J*
-¼..
H>N, :H$.
·*%
n.
'••‘ 'Ί « 9 Y. -I-A....-••to·,X
K N>r' ^-Y---1, ' ‘ 6 K «Λ» (iVb.,8)
Ft
AnM
T Tl
ΥΝ-··ΛΝΥ
H cF^oh
O (IVb.9) / 7
V0
..OH o
UVb.lO) !%R
Ah,
NHn1,1 ..1 γ-'-νΑ,-χν Μ & x H
O X · ''‘NM cr' w,
-139/b.in
Figure GB2552041A_D0158
χ, ’b 13) >.14) .0 /Ύ // η
χ ,χ <? »· ·
H0-S-0 :55
Ο
Figure GB2552041A_D0159
•ΝΗ
Ο^&Η,
OH f Li ..< Ν,..
?ί '>
Ο
Α^χΑ,,Α/
Χγ^ο^Μ 'Μ ^..-Η
.. ^ΐ$>\ .... •s;r νη·5
Figure GB2552041A_D0160
•χ^^ϊχ
ΗχΝ ΧΌ
140 (IVb.lS) to •I έ·ο:!η (IVb. 16) ./ Q >τ° νλ» « J.
.^w· ο
ηο.
;·ιο
ΗΟ ' 4 ί jfef'ί .,-yv.,.0 «ί Λ .□ fV τη η
/X >%Ζ Ο .χ-~
Ν~
Μό
Ό
Μ·»·» f L .Χγ
Μ *
-141(JVcJ)
X?)
Τ)
Τ ο ΗΤ.Α
...,χ
ΗΟ:
ϋΐϊΗ
Τϊ ό
ώΧ^ΟΗ ν-Ο
Ν' 'if
Η I; ο
Η?·;.?
S^·'
X
Τ § Α.,
ΧΤΧ ·'·:·- V ^Τη ,-'ϊΧ-.s cr w
ί.
ο ο
Μ J • υ ί! Τ-5'
$.·?!'
Figure GB2552041A_D0161
(Υγ>ί
Λ,ί
Χόγ
I d χ
-142(IVc.4) (IVeJ) (IVe.6) (iVc.7)
Α:
*4
A,
Ο
HO . g 1 j
Xf' * '^:
:o & z\ '' θ HS T>H
X ^-Ύ bZ™ %<o % A—NH i·
YX> 1—*' p Q K.
0' HiM^C ^“i'
-NH <τ°Α ο Ύ* ... ο ϊΆ
VzAma zvyx i«S
Η,νΆώ <·-Χ Λ'Χ .X.O-L •‘'-'Χ <' -.^- Nq·^ '.£( y/SAxZVNA/ >. .1 ί.ί
Z'-V
-143own 'V „/ f
iJ
ΓΗ / *-
Ύ I f « i
VXx\z
Α·χ°
Β--Ν.
Υ° hUi ο^Ι c,. χ%
Yd3i .4)
ΠΓ
Ο
V ό ·'· .!
'♦'y%AXpX/Aw·’ &Η £:Η ?
κχ ί-Ι ~ NV*Sj
Ο Η kJ j!
x^'^y*·^··’-'-’'’^*''·^*' %x'{--\,
S.» X.· ^V Λ .·*, _ \\ ^·ν'%Χ?>\Ζ WΝχζ*'\Ζ V\
-. --..· X·'
Ν, χ-^Λχ j < ί ί>4«Ο ?*·Τ:Ο
144 [0331] The linker can contain an enzymatically cleavable sugar moiety, for example, a linker comprising structural formula (Va), (Vb), (Vc), (Vd), or (Ve):
Figure GB2552041A_D0162
Figure GB2552041A_D0163
Figure GB2552041A_D0164
-145-
Figure GB2552041A_D0165
or a salt thereof, wherein: q is 0 or 1; r is 0 or 1; X1 is CH2, O or NH; represents the point of attachment of the linker to the immune-stimulatory compound; and * represents the point of attachment to the remainder of the linker.
[0332] Exemplary embodiments of linkers according to structural formula (Va) that can be included in the antibody construct immune-stimulatory compound conjugates described herein can include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):
-146X'Ys».
<Ya
KO' ’&.3>
A) 'foR
OH z% ”X vAx ,o
Τη y
Kcr'Y^»;
•0^ 'ηΑ
K
W p nx^A/\k.A h it , ... t 'N\$7^’'Xx<\^''’^^.<-’‘^\s·'''· '^χ $η t N·· < > <Λ
H A
Κ'Ά .<> k OtH ·' 'γ*’ t
oh yPK •h·· /% o i?
,O, κσi T <> £ / I /χτ 1 %ΧΚχίκ% ys^K/ “ &
V N
Η H
147· u 51 fe
A '··· ,...Ii
HO
i>\ \<ϊ·
3Uf
$ I
HO* χ»Χ V.,. i OH
H f
ft $ v\
W ^.. H £>·'
V
Ii
Figure GB2552041A_D0166
G «S\ u /) faif'v < JS-··' '<& ·.· V* $ 'X
S. /
A
OX
OH fo%: Ο o 0
Α,,κνγχ,ΑΑ'^Λ^ I H H 1 # . ,;Ογ'\·,, rtO I 0H
OH f'l oft z\ v
f ow
148 (Va ’Ό
Figure GB2552041A_D0167
\,s.
nA
Λο ιοί ον ο o 'Α-».
is I I si / X kg*' xy' ^0K L GK x\>
0¼
Wli /V
H <r k\\>.,
I.
\$u v$> 0.^.0·•'X
GG:< /S 0
YsY k JS\ ή -A
SS <\ hct γ
OH
149 [0333] Exemplary embodiments of linkers according to structural formula (Vb) that may be included in the antibody construct immune-stimulatory compound conjugates described herein include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):
Vb, I)
HOiC*
HC
«.•Xs v~V / 1 sArt O' V
OH $,. N SSS'^K \,VSX
Hci OH o
In
Y SS Λ*' d
-150χ.·*·
VftJ)
..'•'φ § v
Y
Η&ν> Ζ\Ζ i
£ ϊθ γ w /V
OR
VM)
...·„© R**
Ϋ R <y V.S Χ \<j ?°>Η j
Ο ΟΗ·\>* f//' < '1 i < <Ύ c\k <ζ· /a sx y Ύ V
OH / .«OM^ ii \ :£$· k<
/ /Z\
Ybz)
/..
\ .···' W
HO
A 1 </
V ho^/1 <ΊΜ AS88& v ./ ix Ζ/χχ. λ .V/ Y //
Λ>
//
Νίί\
V 1). ft) ,'Ά A*
Υ'Ν ,/Ά'χ/ ! // X Ζ .Ζ λ' ίV /)Η
Λ*/·.·. /·'
Λ4?
I /-^/// k ·ϊ„ λ'α' ν.« <
151
Figure GB2552041A_D0168
Figure GB2552041A_D0169
o
J
Figure GB2552041A_D0170
/ \ . / \ (Vb.9)
1 •*'X
v> 0H
ffW Ογ··χ^0Η
9 \ .,···· i un
s jt ,-ν'···'· %a\\{ ·: \? \
X 1
ί 's
Sssi 0 /
<0
/ 4 k X x, <.<·<>
152·
O'% /hie
0,,, ,. N .... .q '' H Cfo ..,,·-% tx > W w ,Zx, <*0R HO Ί
OH 6 [0334] Exemplary embodiments of linkers according to structural formula (Vc) that may be included in the antibody construct immune-stimulatory compound conjugates described herein include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):
HOsx./
Λ*ς<%Η o
fr °V'\ 'C h fr X
... >E£-NvX-X
HO
OH
HO* fr A. fr >^o ·* <·<
-153-
Η <χ Ζ ί ζ*<
ς Aoji
(Vc?) ζΚ ι 1 K<ZAg ,Ζκν. Ζ* :’ Χ\Ζ*
\J·
aAa * \ζ
w
H α/Η
V A«\
V A
AH s ZWXAM ./N,·.^ >·> >·>· V. > A
A V
X'-'SSv
Ί ι z
ΚΛσ
Ζ*
Η' b X'
ΖΧ
W ι Ο
Ο «
A ••Ν' >
?>'
AV
Η ., HQA θ'
ZaG
Figure GB2552041A_D0171
ην>α^νΑ
?ζ
-154α
Υ. Η y- >ύ·» >, .·χ ν s
YRY
I 1
0Η
Μ. ν0 .· χ- \.·.
HR' Ν* ζχ.
Α.·. .0·. ,<··'\ ,.-χ HR ΗίΆΗ
Xy X\->' Nfr Yy5 * χ'x'\y’
HR’’ χ····' Υ.,· ϊ
A
Y^^^^YR^YY^yRR Υ%Η $> Ζί
ΧΫ
Α<<\ ·?Υ
AYyj*^ ^qYYxY ιη ro^z Ύ
JOR ,>-χ .λχ .< >
/ 0-VY υν^ ,ΖΥχ ν\\ tU-w ,>Υ;Ζ A χ3αζ η, ό
155
A
€.9)
Hri
I fo-z
Zss* .,.·ΖΝχΧνΧ' κσ >'Αχ.
OH
ΖίΑ
Figure GB2552041A_D0172
[0335] Exemplary embodiments of linkers according to structural formula (Vd) that may be included in the antibody construct immune-stimulatory compound conjugates described herein include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):
-156(W.h (Vd ;n <V<1 A <W.4i p
•'•y
I i 0 s
jW.
i\
X >< '1 V ,.. tw^/% %·:·/ Ά \ ,A»^''' l 1 x'Xs4''' ?Xsi' /As
Vi ii ν y-v •§3 jSXXs .v.
z Xv v '4 / HO.
χ -,,.,
V - - ' 4..
<X,A'V •Χχ.' )
:\ / $£xw.«· ssZ Vv
OH \ /‘A / yssss^ f V,Z 7^0i
-. .-'X xy 'Xx»'—-X
Η®
Tv xx^_ f >» pd 4.../ 1 \ssX>X
V Λ'Χ.χ. ' Αν··· χ<·
X
A.
$τ·χ\·χ.
{Χ,'χί
Vi >*
A-T >«HH
V.
/V
157·
Figure GB2552041A_D0173
(Vd.6)
Figure GB2552041A_D0174
[0336] Exemplary embodiments of linkers according to structural formula (Ve) that may be included in the antibody construct immune-stimulatory compound conjugates described herein include the linkers illustrated below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct):
Figure GB2552041A_D0175
-158γ.:η
3ίί
Ν,Α [·>
ώ!^·.. .''“χ.. ,-ΛΧχ .νΖ'χ, .> Ν χ,. χ,.
a J δ iJ ί
ΟΗ
J,
ΧΙΟ’ •’«ij»'” 'ijSS'.i \555ϊ/ [0337] Although cleavable linkers can provide certain advantages, the linkers comprising the conjugate described herein need not be cleavable. For non-cleavable linkers, the immune-stimulatory compound release may not depend on the differential properties between the plasma and some cytoplasmic compartments. The release of the immune-stimulatory compound can occur after internalization of the antibody construct immune-stimulatory compound conjugate via antigenmediated endocytosis and delivery to lysosomal compartment, where the antibody construct can be degraded to the level of amino acids through intracellular proteolytic degradation. This process can release a immune-stimulatory compound derivative, which is formed by the immune-stimulatory compound, the linker, and the amino acid residue to which the linker was covalently attached. The immune-stimulatory compound derivative from antibody construct immune-stimulatory compound conjugates with non-cleavable linkers can be more hydrophilic and less membrane permeable, which can lead to less bystander effects and less nonspecific toxicities compared to antibody construct immune-stimulatory compound conjugates with a cleavable linker. Antibody construct immunestimulatory compound conjugates with non-cleavable linkers can have greater stability in circulation than antibody construct immune-stimulatory compound conjugates with cleavable linkers. Noncleavable linkers can be alkylene chains, or can be polymeric, such as, for example, based upon polyalkylene glycol polymers, amide polymers, or can include segments of alkylene chains, polyalkylene glycols and/or amide polymers. The linker can contain a polyethylene glycol segment having from 1 to 6 ethylene glycol units.
-159[0338] The linker can be non-cleavable in vivo, for example, a linker according to the formulations below:
Figure GB2552041A_D0176
Figure GB2552041A_D0177
or salts thereof, wherein: Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate; Rx is a moiety including a functional group capable of covalently linking the linker to an antibody construct;
and r represents the point of attachment of the linker to the immune-stimulatory compound. [0339] Exemplary embodiments of linkers according to structural formula (Vla)-(VId) that may be included in the conjugates described herein include the linkers illustrated below (as illustrated, the /
linkers include a group suitable for covalently linking the linker to an antibody construct, and ' represents the point of attachment to an immune-stimulatory compound):
-160ο
Ο
Η ,ί/'
Ο
ο •X.''
Η \ζ''Χυχ
Xi ο
Figure GB2552041A_D0178
Η %/ΧΝνζ\
Ο ,Λ
Τά •Μ .<·<· χ η
Figure GB2552041A_D0179
ό α
Α .-·%χ \\.
Ο
-161-
Figure GB2552041A_D0180
[0340] Attachment groups that are used to attach the linkers to an antibody can be electrophilic in nature and include, for example, maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, haloformates, acid halides, alkyl, and benzyl halides such as haloacetamides. There are also emerging technologies related to self-stabilizing maleimides and bridging disulfides that can be used in accordance with the disclosure.
[0341] One example of a self-stabilizing maleimide group that hydrolyzes spontaneously under antibody construct conjugation conditions to give an antibody construct immune-stimulatory compound conjugate species with improved stability is depicted in the schematic below. Thus, the maleimide attachment group is reacted with a sulfhydryl of an antibody construct to give an intermediate succinimide ring. The hydrolyzed form of the attachment group is resistant to deconjugation in the presence of plasma proteins.
Ο •'Αχ.
3Z,-<
y-rzX >
/'-Nil
X ,LX t
! N™* o
Leads s'.' “&&& iass* war Sims \ p <i- >
•χ ;
z )
Crintsjre· sarcailiSNis'
m.Ai} \ v, 1 v rix-j &
KN”’*,.
ν’-'’ \T O Ci rMT
OH
UN
-162[0342] A method for bridging a pair of sulfhydryl groups derived from reduction of a native hinge disulfide bond has been disclosed and is depicted in the schematic below. An advantage of this methodology is the ability to synthesize homogenous DAR4 antibody construct immune-stimulatory compound conjugates by full reduction of IgGs (to give 4 pairs of sulfhydryls) followed by reaction with 4 equivalents of the alkylating agent. Antibody construct immune-stimulatory compound conjugates containing bridged disulfides are also claimed to have increased stability.
Ta—a-s—
Figure GB2552041A_D0181
[0343] Similarly, as depicted below, a maleimide derivative that is capable of bridging a pair of sulfhydryl groups has been developed.
Figure GB2552041A_D0182
[0344] The attachment moiety can contain the following structural formulas (Vila), (Vllb), or (Vile):
-163fib)
Figure GB2552041A_D0183
io $Vhbl
VuA <ί χ &
> Ύ
Figure GB2552041A_D0184
R:* or salts thereof, wherein: Rq is H or-O-(CH2CH2O)n-CH3; x is 0 or 1; y is 0 or 1; G2 isCH2CH2CH2SO3H or-CH2CH2O-(CH2CH2O)n-CH3; Rw is-O-CH2CH2SO3H or-NH(CO)CH2CH2O-(CH2CH2O)i2-CH3; and * represents the point of attachment to the remainder of the linker.
[0345] Exemplary embodiments of linkers according to structural formula (Vila) and (Vllb) that can be included in the antibody construct immune-stimulatory compound conjugates described herein can include the linkers illustrated below (as illustrated, the linkers can include a group suitable for covalently linking the linker to an antibody construct):
-164-
Figure GB2552041A_D0185
165
χ.\' (VIM)
V·£ >··' ?
/yV'/
Λ-χΥ..
TV4 A. ' >„<?
A-S^Ay'-Ns, (VUb .4 ,A ,,4 44 =4 ,<<5X '4-..
f sA
K4, 4'Τ’
X
V
TH
Uki * II Hhi-’X V 4 4 H y fs i x /'., 'ik ff λ
Sf”-
Figure GB2552041A_D0186
XT * V A > T ί
Figure GB2552041A_D0187
«X * A V
HHl xs,.
Ίί 0
V Tf H
HN ., 4 $ 44 T , 4X'
V166
Si ' fXs u
Figure GB2552041A_D0188
Figure GB2552041A_D0189
Figure GB2552041A_D0190
Figure GB2552041A_D0191
\ Vx·' \s·· Χχ· rib)
Ν >
Figure GB2552041A_D0192
6κ 6η
-167
μ'ν> μ~< I /'04 >
'. 4
7’χΖ%
0Η <3Η ΧΥΗ
Hi ii Η
5*χ viib.si < 0
ΊΓ ο ···' / U-N I
Figure GB2552041A_D0193
ί0
J *0Η
Ύ^/
OH ΟΗ
Η* Υ kA ,' ' A ν >
[0346] Exemplary embodiments of linkers according to structural formula (Vile) that can be included in the antibody construct immune-stimulatory compound conjugates described herein can include the linkers illustrated below (as illustrated, the linkers can include a group suitable for covalently linking the linker to an antibody construct):
Figure GB2552041A_D0194
-168
Ι^ΖΝΖν%/ν*%Ζ if jiirt 'w <.. . » ί « ί 11 i >*% § f f <«.··
Y « 1 V <.i:5?\ x·'' <:rt azs/sazs/sz»
ilk JI *-$· ξζ
Z-.Z .·:> \<
Z'^z^ is' *·»*' f
<? 1 $z
Ζ'Ζ.ϊ
-169jlv.is •ri:
H ~ S ·<·' « <.x Λύ ,.k .X Υ. %.·*χ. .&' r*v γ Y Y | i j; h i I t r\ a
A y'\-v ^x^-'x·^ ,0
X- ><
\ bo <\ He X
LOsx./
V ,Z' οά kA
OH «Π
UH xr h</ b
L
FIT & . a/ fox b on
U'-··
Figure GB2552041A_D0195
©«sA
C ί
V '<
<\ >. ν' \.i \.>'\x··
Figure GB2552041A_D0196
HH
Figure GB2552041A_D0197
J ? Ή
Conjugates [0347] A composition as described herein can be a conjugate. A conjugate can comprise an antibody construct, an immune-stimulatory compound, and a linker. A conjugate can comprise an antibody construct, a pattern recognition receptor (PRR) agonist, and a linker. A conjugate can comprise an antibody construct, a pattern-associated molecular pattern (PAMP) molecule, and a linker. A conjugate can comprise an antibody construct, a damage-associated molecular pattern (DAMP) molecule, and a linker. A conjugate can comprise an antibody construct, a STING agonist, and a linker. A conjugate can comprise an antibody construct, a toll-like receptor agonist molecule, and a
-170linker. A conjugate can comprise an antibody construct, imiquimod, and a linker. A conjugate can comprise an antibody construct, S-27609, and a linker. A conjugate can comprise an antibody construct, CL307, and a linker. A conjugate can comprise an antibody construct, resiquimod, and a linker. A conjugate can comprise an antibody construct, gardiquimod, and a linker. A conjugate can comprise an antibody construct, UC-IV150, and a linker. A conjugate can comprise an antibody construct, KU34B, and a linker. A conjugate can comprise an antibody construct, motolimod, and a linker. A conjugate can comprise an antibody construct, VTX-1463, and a linker. A conjugate can comprise an antibody construct, GS-9620, and a linker. A conjugate can comprise an antibody construct, GSK2245035, and a linker. A conjugate can comprise an antibody construct, TMX-101, and a linker. A conjugate can comprise an antibody construct, TMX-201, and a linker. A conjugate can comprise an antibody construct, TMX-202, and a linker. A conjugate can comprise an antibody construct, isatoribine, and a linker. A conjugate can comprise an antibody construct, AZD8848, and a linker. A conjugate can comprise an antibody construct, MEDI9197, and a linker. A conjugate can comprise an antibody construct, 3M-051, and a linker. A conjugate can comprise an antibody construct, 3M-852, and a linker. A conjugate can comprise an antibody construct, 3M-052, and a linker. A conjugate can comprise an antibody construct, 3M-854A, and a linker. A conjugate can comprise an antibody construct, S-34240, and a linker. A conjugate can comprise an antibody construct, CL663, and a linker. A conjugate can comprise an antibody construct, KIN1148, and a linker. A conjugate can comprise an antibody construct, SB-9200, and a linker. A conjugate can comprise an antibody construct, KIN-100, and a linker. A conjugate can comprise an antibody construct, ADU-S100, and a linker. A conjugate can comprise an antibody construct, KU34B, and a linker. An antibody construct of any of the conjugates described herein can have a modified Fc domain of the antibody construct. The modified Fc domain can comprise a substitution at more than one amino acid residue such as at 5 different amino acid residues including
L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including S239D/I332E, or as at 3 different amino acid residues including S298A/E333A/K334A. The numbering of amino acids residues described herein can be according to the EU index as in Kabat.
[0348] A conjugate can comprise an antibody construct, a targeting binding domain, an immunestimulatory compound, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, a pattern recognition receptor (PRR) agonist, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, a pattern-associated molecular pattern (PAMP) molecule, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, a damage-associated molecular pattern (DAMP) molecule, and a linker. A conjugate can
-171comprise an antibody construct, a targeting binding domain, a STING agonist, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, a toll-like receptor agonist molecule, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, imiquimod, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, S-27609, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, CL307, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, resiquimod, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, gardiquimod, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, UC-IV150, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, motolimod, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, VTX-1463, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, GS-9620, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, GSK2245035, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, TMX-101, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, TMX-201, and a linker.
A conjugate can comprise an antibody construct, a targeting binding domain, TMX-202, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, isatoribine, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, AZD8848, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, MEDI9197, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, 3M-051, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, 3M-852, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, 3M-052, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, 3M-854A, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, S-34240, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, CL663, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, KIN1148, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, SB-9200, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, KIN-100, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, ADU-S100, and a linker. A conjugate can comprise an antibody construct, a targeting binding domain, KU34B, and a linker. An antibody construct of any of the conjugates described herein can have a modified Fc domain of the antibody construct. The modified Fc domain can comprise a substitution at more than one amino acid residue such as at 5 different amino acid
-172residues including L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including S239D/I332E, or as at 3 different amino acid residues including S298A/E333A/K334A. [0349] The linker can be a linker as described herein. A linker can be cleavable, non-cleavable, hydrophilic, or hydrophobic. A cleavable linker can be sensitive to enzymes. A cleavable linker can be cleaved by enzymes such as proteases. A cleavable linker can be a valine-citrulline or a valinealanine linker. A valine-citrulline or valine-alanine linker can contain a pentafluorophenyl group. A valine-citrulline or valine-alanine linker can contain a succimide group. A valine-citrulline or valinealanine linker can contain a PABA group. A valine-citrulline or valine-alanine linker can contain a PABA group and a pentafluorophenyl group. A valine-citrulline or valine-alanine linker can contain a PABA group and a succinimide group. A non-cleavable linker can be protease insensitive. A noncleavable linker can be maleimidocaproyl linker. A maleimidocaproyl linker can comprise Nmaleimidomethylcyclohexane-1 -carboxylate. A maleimidocaproyl linker can contain a succinimide group. A maleimidocaproyl linker can contain pentafluorophenyl group. A linker can be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. A linker can be a maleimide-PEG4 linker. A linker can be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. A linker can be a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules. A linker can contain maleimides linked to polyethylene glycol molecules in which the polyethylene glycol can allow for more linker flexibility or can be used lengthen the linker. A linker can be a (maleimidocaproyl)-(valine-citrulline)-(paraaminobenzyloxycarbonly)-(NH2) linker. A linker can be a THIOMAB linker. A THIOMAB linker can be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonly)-(NH2) linker. A linker can also be an alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides, or aminobenzylcarbamates. A linker can contain a maleimide at one end and an N-hydroxysuccinimidyl ester at the other end. A linker can contain a lysine with an N-terminal amine acetylated, and a valine-citrulline cleavage site. A linker can be a link created by a microbial transglutaminase, wherein the link is created between an aminecontaining moiety and a moiety engineered to contain glutamine as a result of the enzyme catalyzing a bond formation between the acyl group of a glutamine side chain and the primary amine of a lysine chain. A linker can contain a reactive primary amine. A linker can be a Sortase A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG (SEQ ID NO: 21) recognition motif to an N-terminal GGG motif to regenerate a native amide bond. The linker created can therefore link a moiety attached to the LXPTG (SEQ ID NO: 21) recognition motif with a moiety attached to the
-173N-terminal GGG motif. A linker can be a link created between an unnatural amino acid on one moiety reacting with oxime bond that was formed by modifying a ketone group with an alkoxyamine on another moiety. A moiety can be an antibody construct. A moiety can be a targeting binding domain. A moiety can be an antibody. A moiety can be an immune-stimulatory compound.
[0350] The antibody construct can be as described herein. The antibody construct can be an antitumor antigen antibody construct. The antibody construct can be an anti-tumor antigen antibody. An antigen recognized by the antibody construct can be CD5, CD19, CD20, CD25, CD37, CD30,
CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC,HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, pl85HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin BI, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, or Fos-related antigen 1. The antibody construct can recognize an antigen that can be expressed on a cell. The antibody construct can recognize an antigen that can be expressed by a cell. The antibody construct can recognize an antigen that can be expressed in the context of a Major Histocompatibility Complex. The antibody construct can recognize an antigen that can stimulate activity of a cell. The antibody construct can recognize an antigen that can stimulate an immune response. The antibody construct can recognize an antigen that can reduce an immune response. The antibody construct can recognize an antigen can reduce activity of a cell. The antibody construct can recognize an antigen that can be expressed on an immune cell. The antibody construct can recognize an antigen that can be expressed by an immune cell. The antibody construct can recognize an antigen that can be in the context of a Major Histocompatibility Complex. The antibody construct can recognize an antigen on a cell wherein the antigen can be involved in stimulating activity of a cell. The antibody construct can recognize an antigen on an immune cell that can be involved in the costimulation of an immune cell. The antibody construct can recognize an antigen on an immune cell that can be involved in the costimulation of an immune cell
-174during an immune response. The antibody construct can recognize a receptor. The antibody construct can recognize a receptor on a cell. The antibody construct can recognize a receptor ligand. The antibody construct can recognize a receptor on a cell wherein the receptor can be involved in stimulating activity of a cell. The antibody construct can recognize a receptor on an immune cell.
The antibody construct can recognize a receptor on an immune cell that can be involved in stimulating activity of an immune cell. The antibody construct can recognize a receptor on an immune cell that can be involved in the costimulation of an immune cell. The antibody construct can recognize a receptor on an immune cell that can be involved in the costimulation of an immune cell during an immune response. The antibody construct can recognize an antigen that can be expressed on an immune cell and that can stimulate activity of an immune cell. The antibody construct can recognize an antigen that can be expressed on an immune that can reduce activity of an immune cell. The antibody construct can be an anti-CD40 antibody. The antibody construct can comprise a light chain of an SBT-040 antibody. The antibody construct can comprise an SBT-040-G1WT heavy chain. The antibody construct can comprise an SBT-040-G1VLPLL heavy chain. The antibody construct can comprise an SBT-040-G1DE heavy chain. The antibody construct can comprise an SBT-040-G1AAA heavy chain. The antibody construct can comprise an SBT-040-CDR sequence. The antibody construct can be capable of recognizing a single antigen. The antibody construct can be capable of recognizing two or more antigens. The Kd for binding of an antigen-binding domain of an antibody construct immune-stimulatory compound conjugate to an antigen in the presence of an immune-stimulatory compound can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the Ka for binding of the antigen binding domain to the antigen of an antibody construct in the absence of the immune-stimulatory compound. The Kd for binding of an antigen-binding domain of an antibody construct immune-stimulatory compound conjugate to an antigen in the presence of the immune-stimulatory compound can be less than 10 nM. The Kd for binding of an antigen-binding domain of an antibody construct immune-stimulatory compound conjugate to an antigen in the presence of the immune-stimulatory compound can be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM. [0351] An antibody construct can further comprise a targeting binding domain. A targeting binding domain of an antibody construct can recognize an antigen. For example, an antigen can be expressed on an immune cell. As another example, an antigen can be expressed by a tumor or cancer cell. An
-175antigen can be a peptide or fragment thereof. An antigen can be expressed on an antigen-presenting cell. An antigen can be expressed on a dendritic cell, a macrophage, or a B cell. An antigen can be CD40 and a targeting binding domain can recognize a CD40 antigen. An antigen can be a tumor antigen and a targeting binding domain can recognize a tumor antigen. A targeting binding domain of an antibody construct can be a CD40 agonist. A targeting binding domain of an antibody construct can bind to a tumor antigen.
[0352] The antibody construct can have an Fc domain that can bind to an FcR when linked to an immune-stimulatory compound. The antibody construct can have an Fc domain that can bind to an FcR to initiate FcR-mediated signaling when linked to an immune stimulatory compound. The antibody construct can bind to its antigen when linked to an immune-stimulatory compound. The antibody construct can bind to its antigen when linked to an immune-stimulatory compound and the Fc domain of the antibody construct can bind to an FcR when linked to an immune-stimulatory compound. The antibody construct can bind to its antigen when linked to an immune-stimulatory compound and the Fc domain of the antibody can bind to an FcR to initiate FcR-mediated signaling when linked to an immune stimulatory compound. The Fc domain linked to an immune-stimulatory compound can be a modified Fc domain. The modified Fc domain can comprise a substitution at more than one amino acid residue such as at 5 different amino acid residues including
F235V/F243F/R292P/Y300F/P396F, as at 2 different amino acid residues including S239D/I332E, or as at 3 different amino acid residues including S298A/E333 A/K334A. The Ka for binding of an Fc domain to a Fc receptor when the Fc domain is linked to an immune-stimulatory compound can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the Ka for binding of the Fc domain to the Fc receptor in the absence of the immune-stimulatory compound. The Ka for binding of an Fc domain to an Fc receptor when linked to an immunestimulatory compound can be less than 10 nM. The Ka for binding of an Fc domain to an Fc receptor when linked to an immune-stimulatory compound can be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM.
[0353] The PRR agonist can be a toll-like receptor agonist. The toll-like receptor agonist can be a TFR1 agonist, a TFR2 agonist, a TFR3 agonist, a TFR4 agonist, a TFR5 agonist, a TFR6 agonist, a TFR7 agonist, a TFR8 agonist, a TFR9 agonist, a TFR10 agonist, a TFR11 agonist, a TFR12
-176agonist or a TLR13 agonist. The toll-like receptor agonist can activate two or more TLRs. The PAMP molecule can be a RIG-I agonist.
[0354] A conjugate can be formed by a linker that can connect an antibody construct to a PRR. A conjugate can be formed by a linker that can connect an antibody construct to a PAMP molecule. A conjugate can be formed by a linker that can connect an antibody construct and a DAMP molecule.
A conjugate can be formed by a linker that can connect an antibody construct to a PRR, and a linker that can connect an antibody construct and a targeting binding domain. A conjugate can be formed by a linker that can connect an antibody construct to a PAMP molecule, and a linker that can connect an antibody construct and a targeting binding domain. A conjugate can be formed by a linker that can connect an antibody construct and a DAMP molecule, and a linker that can connect an antibody construct and a targeting binding domain.
[0355] A linker can be connected to an antibody construct by a direct linkage between the antibody construct and the linker. A linker can be connected to an anti-CD40 antibody construct by a direct linkage between the anti-CD40 antibody construct and the linker. A linker can be connected to an anti-CD40 antibody by a direct linkage between the anti-CD40 antibody and the linker. A linker can be connected to an anti-tumor antigen antibody construct by a direct linkage between the anti-tumor antigen antibody construct and the linker. A linker can be connected to an anti-tumor antigen antibody by a direct linkage between the anti-tumor antigen antibody and the linker. A direct linkage can be a covalent bond. For example, a linker can be attached to a terminus of an amino acid sequence of an antibody construct, or could be attached to a side chain modification to the antibody construct, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a nonnatural amino acid residue, or glutamic acid residue. An attachment can be via any of a number of bonds, for example but not limited to, an amide bond, an ester bond, an ether bond, a carbonnitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. A linker can have at least one functional group, which can be linked to the antibody. Non-limiting examples of the functional groups can include those which form an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond, such functional groups can be, for example, amino groups; carboxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; carbonates; carbonyl functionalities bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups. A linker can be connected to an antibody construct at a hinge cysteine. A linker can be connected to an antibody construct at a light chain constant domain lysine. A linker can be connected to an antibody construct at an engineered cysteine in the light chain. A linker can be connected to an antibody construct at an engineered light chain glutamine. A linker can
-177be connected to an antibody construct at an unnatural amino acid engineered into the light chain. A linker can be connected to antibody construct at an unnatural amino acid engineered into the heavy chain. Amino acids can be engineered into an amino acid sequence of a composition as described herein, for example, a linker of a conjugate. Engineered amino acids can be added to a sequence of existing amino acids. Engineered amino acids can be substituted for one or more existing amino acids of a sequence of amino acids. A linker can be conjugated to antibody construct via a sulfhydryl group. A linker can be conjugated to an antibody construct via a primary amine. A linker can be a link created between an unnatural amino acid on an antibody construct reacting with oxime bond that was formed by modifying a ketone group with an alkoxyamine on an immune-stimulatory compound. When a linker is connected to an antibody construct at the sites described herein, an Fc domain of the antibody construct can bind to Fc receptors. When a linker is connected to an antibody construct at the sites described herein, the antigen binding domain of the antibody construct can bind its antigen. When a linker is connected to an antibody construct at the sites described herein, a targeting binding domain of said antibody construct can bind its antigen.
[0356] An antibody with engineered reactive cysteine residues (THIOMAB) can be used to link a targeting binding domain to the antibody. A linker can connect an antibody construct to a targeting binding domain via Sortase A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG (SEQ ID NO: 21) recognition motif to an N-terminal GGG motif to regenerate a native amide bond. The linker created can therefore link an antibody construct attached to the LXPTG (SEQ ID NO: 21) recognition motif with a targeting binding domain attached to the Nterminal GGG motif. A targeting binding domain can be connected to a linker by a direct linkage. A direct linkage can be a covalent bond. For example, a linker can be attached to a terminus of an amino acid sequence of a targeting binding domain, or could be attached to a side chain modification to the targeting binding domain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid residue. An attachment can be via any of a number of bonds, for example but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. A linker can have at least one functional group, which can be linked to the targeting binding domain. Non-limiting examples of the functional groups can include those which form an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond, such functional groups can be, for example, amino groups; carboxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; carbonates; carbonyl functionalities bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups.
-178Amino acids can be engineered into an amino acid sequence of the targeting binding domain. Engineered amino acids can be added to a sequence of existing amino acids. Engineered amino acids can be substituted for one or more existing amino acids of a sequence of amino acids. A linker can be conjugated to a targeting binding domain via a sulfhydryl group. A linker can be conjugated to a targeting binding domain via a primary amine. A targeting binding domain can be conjugated to the C-terminal of an Fc domain of an antibody construct.
[0357] An antibody with engineered reactive cysteine residues (THIOMAB) can be used to link an immune-stimulatory compound to the antibody. A linker can connect an antibody construct to an immune-stimulatory compound via THIOMAB linker. A linker can connect an antibody construct to an immune-stimulatory compound via Sortase A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an LXPTG (SEQ ID NO: 21) recognition motifto an N-terminal GGG motif to regenerate a native amide bond. The linker created can therefore link an antibody construct attached the LXPTG (SEQ ID NO: 21) recognition motif with an immune-stimulatory compound attached to the N-terminal GGG motif. A linker can be a link created between an unnatural amino acid on an antibody construct reacting with oxime bond that was formed by modifying a ketone group with an alkoxyamine on an immune-stimulatory compound. The immune-stimulatory compound can comprise one or more rings selected from carbocyclic and heterocyclic rings. The immune-stimulatory compound can be covalently bound to a linker by a bond to an exocyclic carbon or nitrogen atom on said immune-stimulatory compound. A linker can be conjugated to an immunestimulatory compound via an exocyclic nitrogen or carbon atom of an immune-stimulatory compound. A linker can be connected to a STING agonist, for example:
Figure GB2552041A_D0198
Figure GB2552041A_D0199
-1790
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X
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Figure GB2552041A_D0200
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H0, ?
Figure GB2552041A_D0201
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.,0-0,.
O ··<
Figure GB2552041A_D0202
ri .ri.
ri ri if .ri, ί ΙΑ A 'ri :
or
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ri' .0-0.
Figure GB2552041A_D0203
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O.....K ri .,0, ri ,ri, i 0 ri iri >X. ·>· 0H ri
Hri.
ri ^00mhfeA0O4-e-0<S'-riH0p-0P [0358] A linker agonist complex can dissociate under physiological conditions to yield an active agonist.
[0359] A linker can be connected to a PRR agonist by a direct linkage between the PRR agonist and the linker. A linker can be connected to a PAMP molecule by a direct linkage between the PAMP molecule and the linker. A linker can be connected to a toll-like receptor agonist by a direct linkage between the toll-like receptor agonist and the linker.
-180[0360] Examples of toll-like receptor agonists connected to a linker in a manner able to release an active toll-like receptor agonist under physiologic condition can include:
Figure GB2552041A_D0204
NHS-PEG5-KU34b
Figure GB2552041A_D0205
Maleimide-Val-Ala-PABA-KU34b
Figure GB2552041A_D0206
Figure GB2552041A_D0207
NHS-Val-Ala-PABA-KU34b [0361] Examples of RIG-I agonists connected to a linker in a manner able to release an active tolllike receptor agonist under physiologic conditions can include:
-181-
Figure GB2552041A_D0208
Figure GB2552041A_D0209
NHS-PEG5-KIN700
Figure GB2552041A_D0210
Maleimide-Val-Ala-PABC-KIN700
Figure GB2552041A_D0211
Figure GB2552041A_D0212
NHS-Va l-Ala-PABC-KIN700 [0362] A linker can be connected to a DAMP molecule by a direct linkage between the DAMP molecule and the linker. A direct linkage can be a covalent bond. For example, a linker can be attached to a terminus of an amino acid sequence of an antibody, or could be attached to a side chain modification to the antibody, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, -182aspartic acid, a non-natural amino acid residue, or glutamic acid residue. An attachment can be via any of a number of bonds, for example but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. A linker can have at least one functional group, which can be linked to the antibody construct. Non-limiting examples of the functional groups can include those which form an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond, such functional groups can be, for example, amino groups; carboxyl groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones; carbonates; carbonyl functionalities bonded to leaving groups such as cyano and succinimidyl and hydroxyl groups.
[0363] An ATAC can be formed by conjugating a noncleavable maleimide-PEG4 linker containing a succinimide group with an immune-stimulatory compound. For example, an ATAC can be N-((4amino-l-(2-hydroxy-2-methylpropyl)-lE[-imidazo[4,5-c]quinolin-2-yl)methyl)-l-(3-(2,5-dioxo-2,5dihydro-ΙΗ-pyrrol-1 -yl)propanamido)-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (ATAC 11); N-(5-(2-amino-3-pentylquinolin-5-yl)pentyl)-l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC 12); l-(3-(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)propanamido)-N-(3-pentylquinolin-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC13); 1-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-(l-isobutyl-lHimidazo[4,5-c]quinolin-4-yl)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC14); l-(3-(2,5-dioxo2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-methyl-N-(2-(3-(7-methylbenzo[l,2-d:3,4d']bis(thiazole)-2-yl)ureido)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC15); (S)-1 -(3-(2,5dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-(l-((7-methylbenzo[l,2-d:3,4-d']bis(thiazole)-2yl)amino)-1 -oxo-3-phenylpropan-2-yl)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC 16); N(benzo[d]thiazol-2-yl)-1 -(3 -(2,5-dioxo-2,5-dihydro- ΙΗ-pyrrol-1 -yl)propanamido)-N-((8hydroxyquinolin-7-yl)(4-(trifluoromethoxy)phenyl)methyl)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC17); N-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9-bis(2-amino-6-oxo-lH-purin-9(6H)-yl)5.10.12- trihydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-j][l,3,7,9,2,8]tetraoxadiphosphacyclododecin-3-yl)-l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)3.6.9.12- tetraoxapentadecan-15-amide (ATAC18); N-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9bis(2-amino-6-oxo-lH-purin-9(6H)-yl)-10-hydroxy-5,12-dimercapto-5,12dioxidodecahydrodifuro[3,2-d:3',2'-j][l,3,7,9,2,8]tetraoxadiphosphacyclododecin-3-yl)-l-(3-(2,5dioxo-2,5-dihydro-1 El-pyrrol-1 -yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC 19); N-(9-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-9-(2-amino-6-oxo-lH-purin-9(6H)-yl)-3,5,10,12tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-j][l,3,7,9,2,8]tetra-183oxadiphosphacy clododecin-2-yl)-9H-purin-6-yl)-1 -(3 -(2,5 -dioxo-2,5-dihy dro-1 H-pyrrol-1 yl)propanamido)-3,6,9,12-tetraoxapentadecan-l5-amide (ATAC20); or N-(9((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-9-(2-amino-6-oxo-lH-purin-9(6H)-yl)-3,5,10,12tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-j][l,3,7,9,2,8]tetraoxadiphosphacyclododecin2- yl)-9H-purin-6-yl)-1-(3 -(2,5-dioxo-2,5 -dihydro-1 H-pyrrol-1 -yl)propanamido)-3,6,9,12tetraoxapentadecan-15-amide (ATAC21).
[0364] An ATAC can be formed by conjugating a cleavable valine-alanine or valine-citrulline linker containing a PABA group and a succinimide group with an immune-stimulatory compound. For example, an ATAC can be 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihy dro-ΙΗ-pyrrol-l -yljhexanamido)3- methylbutanamido)propanamido)benzyl ((4-amino-l-(2-hydroxy-2-methyl-propyl)-lHimidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate (ATAC22); 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5dihydro-lH-pyrrol-l-yl)hexanamido)-3-methyl-butanamido)propanamido)benzyl (5-(2-amino-3pentylquinolin-5-yl)pentyl)-carbamate (ATAC23); 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lHpyrrol-l-yl)hexanamido)-3-methylbutan-amido)-5-ureidopentanamido)benzyl-(5-(2-amino-3pentylquinolin-5-yl)pentyl)-carbamate (ATAC24); 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lHpyrrol-1 -yl)hexanamido)-3 -methylbutanamido)-5-ureidopentanamido)benzyl((4-amino-1 -(2hydroxy-2-methylpropyl)- lH-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate TFA salt (ATAC25); 2-(3-{2-[N-Methyl({p-[(S)-2-{(S)-2-[6-(2,5-dioxo-lH-pyrrol-l-yl)hexanoylamino]-3methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino]ethyl}ureido)-7methyl-1,6-dithia-3,8-diaza-as-indacene (ATAC26); 2-{[(8-Hydroxy-7-quinolyl)(ptrifluoromethoxyphenyl)methyl]({p-[(S)-2-{(S)-2-[6-(2,5-dioxo-lH-pyrrol-l-yl)hexanoylamino]-3methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino}-l,3-benzothi azole (ATAC27); (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-lyl)hexanoylamino]-3-methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonylamino)8.17- bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-3,12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa3λ5.12λ5-diphosphatricyclo[ 13.3.0.06,10]octadecane-3,12-dione (ATAC28); (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-lyl)hexanoylamino]-3-methylbutyrylamino}propionylamino]phenyl}methoxycarbonylamino)-8,17bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-3,12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa3X5.12X5-diphosphatricyclo[13.3.0.06,10]octadecane-3,12-dione (ATAC29); (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-lyl)hexanoylamino]-3-methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonylamino)8.17- bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-9-hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa-1843X5.12X5-diphosphatricyclo[13.3.0.06,10]octadecane-3,12-dione (ATAC30); {p-[(S)-2-{(S)-2-[6(2,5-Dioxo-lH-pyrrol-l-yl)hexanoylamino]-3-methylbutyrylamino}-5ureidovalerylamino]phenyl}methyl 9-{(lS,6R,8R,9S,10S,15R,17R,18S)-8-(2-amino-6-oxo-l,9dihydropurin-9-yl)-3,12-dihydroxy-9,18-dihydroxy-3,12-dioxo-2.4.7.11.13.16-hexaoxa-3k5.12X5diphosphatricyclo[13.2.1.06,10]octadec-17-yl}-9a-adenineecarboxylate (ATAC31; l-{6-[({7Amino-3-(2-hydroxy-2-methylpropyl)-3.5.8-triazatricyclo[7.4.0.02,6]trideca-l(9),2(6),4,7,10,12hexaen-4-yl}methyl)-N-ethylamino]-6-oxohexyl}-lH-pyrrole-2,5-dione (ATAC32); l-{[4-({6-[({7Amino-3-(2-hydroxy-2-methylpropyl)-3.5.8-triazatricyclo[7.4.0.02,6]trideca-l(9),2(6),4,7,10,12hexaen-4-yl}methyl)-N-ethylamino]-6-oxohexylamino}carbonyl)cyclohexyl]methyl}-lH-pyrrole2.5- dione (ATAC33); or l-[(4-{[({7-Amino-3-(2-hydroxy-2-methylpropyl)-3.5.8triazatricyclo[7.4.0.02,6]trideca-l(9),2(6),4,7,10,12-hexaen-4-yl}methyl)-N-ethylamino]carbonyl}cyclohexyl)methyl]-lH-pyrrole-2,5-dione (ATAC34).
[0365] An ATAC can be formed by conjugating a noncleavable maleimide-PEG4 linker containing an activated ester such as a pentafluorophenyl group or an N-hydroxysuccinimide group with an immune-stimulatory compound. For example, an ATAC can be pentafluorophenyl 25-(2-amino-3pentylquinolin-5-yl)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC 1); perfluorophenyl 3-((4-amino-l-(2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2yl)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oate (ATAC2); pentafluorophenyl 25(2-amino-3-pentylquinolin-5-yl)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC3); or
2.5- Dioxopyrrolidin-l-yl 3-((4-amino-l-(2-hydroxy-2-methylpropyl)-lH-imidazo-[4,5-c]quinolin-2yl)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3 -azadocosan-22-oate (ATAC4).
[0366] An ATAC can be formed by conjugating a cleavable valine-alanine or valine-citrulline linker containing a PABA group and an activated ester such as a pentafluorophenyl group or an Nhydroxysuccinimde group to an immune-stimulatory compo pound. For example, an ATAC can be
2.5- dioxopyrrolidin-1 -yi 6-(((S)-1 -(((S)-1 -((4-((((5-(2-amino-3-pentylquinolin-5yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 -oxobutan-2yl)amino)-6-oxohexanoate (ATAC5); 2,5-dioxopyrrolidin-1 -yi 7-(((S)-1 -(((S)-1 -((4-(((((4-amino-1 (2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC6); 2,5-dioxopyrrolidin-l-yl 7-(((S)-l-(((S)-l-((4(((((4-amino-1 -(2-hydroxy-2-methylpropyl)-1 H-imidazo[4,5 -c] quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC7); perfluorophenyl 6-(((S)-l-(((S)-1-((4-185((((5-(2-amino-3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxopropan-2yl)amino)-3-methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC8); perfluorophenyl 7-(((S)-l(((S)-1 -((4-(((((4-amino-1 -(2-hydroxy-2-methylpropyl)- lH-imidazo[4,5-c]quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC9); or perfluorophenyl 7-(((S)-l-(((S)-l-((4-(((((4amino-l-(2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC10).
[0367] An antibody construct can comprise an anti-CD40 antibody. An anti-CD40 antibody can comprise two SBT-040-G1WT heavy chains and two light chain from a SBT-040 antibody, which can be referred to as SBT-040-WT or as SBT-040-G1. An anti-CD40 antibody can comprise two SBT-040-G1VLPLL heavy chains and two light chains from a SBT-040 antibody, which can be referred to as SBT-040-VLPLL. An anti-CD40 antibody can comprise two SBT-040-G1DE heavy chains and two light chains from a SBT-040 antibody, which can be referred to as SBT-040-DE. An anti-CD40 antibody can comprise two SBT-040-G1 AAA heavy chains and two light chains from a SBT-040 antibody, which can be referred to as SBT-040-AAA. An anti-CD40 antibody can comprise two IgG2 heavy chains and two light chain from a SBT-040 antibody, which can be referred to as SBT-040-G2.
[0368] A conjugate can comprise SBT-040-WT-ATAC1. A conjugate can comprise SBT-040-WTATAC2. A conjugate can comprise SBT-040-WT-ATAC3. A conjugate can comprise SBT-040WT-ATAC4. A conjugate can comprise SBT-040-WT-ATAC5. A conjugate can comprise SBT040-WT-ATAC6. A conjugate can comprise SBT-040-WT-ATAC7. A conjugate can comprise SBT-040-WT-ATAC8. A conjugate can comprise SBT-040-WT-ATAC9. A conjugate can comprise SBT-040-WT-ATAC10. A conjugate can comprise SBT-040-WT-ATAC11. A conjugate can comprise SBT-040-WT-ATAC12. A conjugate can comprise SBT-040-WT-ATAC13. A conjugate can comprise SBT-040-WT-ATAC14. A conjugate can comprise SBT-040-WT-ATAC15. A conjugate can comprise SBT-040-WT-ATAC16. A conjugate can comprise SBT-040-WT-ATAC17. A conjugate can comprise SBT-040-WT-ATAC18. A conjugate can comprise SBT-040-WTATAC19. A conjugate can comprise SBT-040-WT-ATAC20. A conjugate can comprise SBT-040WT-ATAC21. A conjugate can comprise SBT-040-WT-ATAC22. A conjugate can comprise SBT040-WT-ATAC23. A conjugate can comprise SBT-040-WT-ATAC24. A conjugate can comprise SBT-040-WT-ATAC25. A conjugate can comprise SBT-040-WT-ATAC26. A conjugate can comprise SBT-040-WT-ATAC27. A conjugate can comprise SBT-040-WT-ATAC28. A conjugate
-186can comprise SBT-040-WT-ATAC29. A conjugate can comprise SBT-040-WT-ATAC30. A conjugate can comprise SBT-040-WT-ATAC31. A conjugate can comprise SBT-040-WT-ATAC32. A conjugate can comprise SBT-040-WT-ATAC33. A conjugate can comprise SBT-040-WTATAC34. A conjugate can comprise SBT-040-WT-ATAC43. A conjugate can comprise SBT-040VLPLL-ATAC1. A conjugate can comprise SBT-040-VLPLL-ATAC2. A conjugate can comprise SBT-040-VLPLL-ATAC3. A conjugate can comprise SBT-040-VLPLL-ATAC4. A conjugate can comprise SBT-040-VLPLL-ATAC5. A conjugate can comprise SBT-040-VLPLL-ATAC6. A conjugate can comprise SBT-040-VLPLL-ATAC7. A conjugate can comprise SBT-040-VLPLLATAC8. A conjugate can comprise SBT-040-VLPLL-ATAC9. A conjugate can comprise SBT-040VLPLL-ATAC10. A conjugate can comprise SBT-040-VLPLL-ATAC11. A conjugate can comprise SBT-040-VLPLL-ATAC12. A conjugate can comprise SBT-040-VLPLL-ATAC13. A conjugate can comprise SBT-040-VLPLL-ATAC14. A conjugate can comprise SBT-040-VLPLL-ATAC15. A conjugate can comprise SBT-040-VLPLL-ATAC16. A conjugate can comprise SBT-040-VLPLLATAC17. A conjugate can comprise SBT-040-VLPLL-ATAC18. A conjugate can comprise SBT040-VLPLL-ATAC19. A conjugate can comprise SBT-040-VLPLL-ATAC20. A conjugate can comprise SBT-040-VLPLL-ATAC21. A conjugate can comprise SBT-040-VLPLL-ATAC22. A conjugate can comprise SBT-040-VLPLL-ATAC23. A conjugate can comprise SBT-040-VLPLLATAC24. A conjugate can comprise SBT-040-VLPLL-ATAC25. A conjugate can comprise SBT040-VLPLL-ATAC26. A conjugate can comprise SBT-040-VLPLL-ATAC27. A conjugate can comprise SBT-040-VLPLL-ATAC28. A conjugate can comprise SBT-040-VLPLL-ATAC29. A conjugate can comprise SBT-040-VLPLL-ATAC30. A conjugate can comprise SBT-040-VLPLLATAC31. A conjugate can comprise SBT-040-VLPLL-ATAC32. A conjugate can comprise SBT040-VLPLL-ATAC33. A conjugate can comprise SBT-040-VLPLL-ATAC34. A conjugate can comprise SBT-040-VLPLL-ATAC43.A conjugate can comprise SBT-040-DE-ATAC1. A conjugate can comprise SBT-040-DE-ATAC2. A conjugate can comprise SBT-040-DE-ATAC3. A conjugate can comprise SBT-040-DE-ATAC4. A conjugate can comprise SBT-040-DE-ATAC5. A conjugate can comprise SBT-040-DE-ATAC6. A conjugate can comprise SBT-040-DE-ATAC7. A conjugate can comprise SBT-040-DE-ATAC8. A conjugate can comprise SBT-040-DE-ATAC9. A conjugate can comprise SBT-040-DE-ATAC10. A conjugate can comprise SBT-040-DE-ATAC1E A conjugate can comprise SBT-040-DE-ATAC12. A conjugate can comprise SBT-040-DE-ATAC13.
A conjugate can comprise SBT-040-DE-ATAC14. A conjugate can comprise SBT-040-DEATAC15. A conjugate can comprise SBT-040-DE-ATAC16. A conjugate can comprise SBT-040DE-ATAC17. A conjugate can comprise SBT-040-DE-ATAC18. A conjugate can comprise SBT-187040-DE-ATAC19. A conjugate can comprise SBT-040-DE-ATAC20. A conjugate can comprise SBT-040-DE-ATAC2E A conjugate can comprise SBT-040-DE-ATAC22. A conjugate can comprise SBT-040-DE-ATAC23. A conjugate can comprise SBT-040-DE-ATAC24. A conjugate can comprise SBT-040-DE-ATAC25. A conjugate can comprise SBT-040-DE-ATAC26. A conjugate can comprise SBT-040-DE-ATAC27. A conjugate can comprise SBT-040-DE-ATAC28. A conjugate can comprise SBT-040-DE-ATAC29. A conjugate can comprise SBT-040-DEATAC30. A conjugate can comprise SBT-040-DE-ATAC3E A conjugate can comprise SBT-040DE-ATAC32. A conjugate can comprise SBT-040-DE-ATAC33. A conjugate can comprise SBT040-DE-ATAC34. A conjugate can comprise SBT-040-DE-ATAC43. A conjugate can comprise SBT-040-AAA-ATACE A conjugate can comprise SBT-040-AAA-ATAC2. A conjugate can comprise SBT-040-AAA-ATAC3. A conjugate can comprise SBT-040-AAA-ATAC4. A conjugate can comprise SBT-040-AAA-ATAC5. A conjugate can comprise SBT-040-AAA-ATAC6. A conjugate can comprise SBT-040-AAA-ATAC7. A conjugate can comprise SBT-040-AAAATAC8. A conjugate can comprise SBT-040-AAA-ATAC9. A conjugate can comprise SBT-040AAA-ATAC10. A conjugate can comprise SBT-040-AAA-ATAC1E A conjugate can comprise SBT-040-AAA-ATAC12. A conjugate can comprise SBT-040-AAA-ATAC13. A conjugate can comprise SBT-040-AAA-ATAC14. A conjugate can comprise SBT-040-AAA-ATAC15. A conjugate can comprise SBT-040-AAA-ATAC16. A conjugate can comprise SBT-040- AAA ATAC17. A conjugate can comprise SBT-040-AAA-ATAC18. A conjugate can comprise SBT-040 AAA-ATAC19. A conjugate can comprise SBT-040-AAA-ATAC20. A conjugate can comprise SBT-040-AAA-ATAC2E A conjugate can comprise SBT-040-AAA-ATAC22. A conjugate can comprise SBT-040-AAA-ATAC23. A conjugate can comprise SBT-040-AAA-ATAC24. A conjugate can comprise SBT-040-AAA-ATAC25. A conjugate can comprise SBT-040-AAAATAC26. A conjugate can comprise SBT-040-AAA-ATAC27. A conjugate can comprise SBT-040 AAA-ATAC28. A conjugate can comprise SBT-040-AAA-ATAC29. A conjugate can comprise SBT-040-AAA-ATAC30. A conjugate can comprise SBT-040-AAA-ATAC3E A conjugate can comprise SBT-040-AAA-ATAC32. A conjugate can comprise SBT-040-AAA-ATAC33. A conjugate can comprise SBT-040-AAA-ATAC34. A conjugate can comprise SBT-040-AAAATAC33. A conjugate can comprise SBT-040-AAA-ATAC43. The Ka for binding of the CD40 binding domain of any of these conjugates to CD40 can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times,
-188about 100 times, about 110 times, or about 120 times greater than the Ka for binding of the CD40 binding domain to CD40 in the absence of the immune-stimulatory compound or ATAC. The Ka for binding of the CD40 binding domain of any of these conjugates to CD40 can be less than 10 nM. The Ka for binding of the CD40 binding domain of any of the conjugates to CD40 can be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM. The Ka for binding of the Fc domain of any of the conjugates to an Fc receptor can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the Ka for binding of the Fc domain to the Fc receptor in the absence of the immune-stimulatory compound or ATAC. The Ka for binding of the Fc domain of any of the conjugates to an Fc receptor of an can be less than 10 nM. The Ka for binding of the Fc domain of any of the conjugates to an Fc receptor can be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM.
[0369] In a conjugate, an antibody can be linked to an immune-stimulatory compound in such a way that the antibody can still bind to an antigen and the Fc domain of the antibody can still bind to an FcR. In a conjugate, an antibody construct is linked to an immune-stimulatory compound in such a way that the linking does not interfere with ability of the antigen binding domain of the antibody construct to bind to antigen, the ability of the Fc domain of the antibody construct to bind to an FcR, or FcR-mediated signaling resulting from the Fc domain of the antibody construct from binding to an FcR. In a conjugate, an immune-stimulatory compound can be linked to an antibody construct in such a way the linking does not interfere with the ability of the immune-stimulatory compound to bind to its receptor. A conjugate can produce stronger immune stimulation and a greater therapeutic window than components of the conjugate alone. In an anti-CD40 antibody linked to a TFR agonist conjugate, the combination of CD40 agonism, TLR agonism, and an accessible Fc domain of the anti-CD40 antibody to allow FcR-mediated signaling can produce stronger immune stimulation and a greater therapeutic window than the CD40 agonism, TLR agonism, or the FcR-mediated signaling alone.
Methods of Synthesis of Antibody Construct Immune-Stimulatory Compound Conjugate
Components
Synthesis of Immune-Stimulatory Compounds
-189[0370] An immune stimulatory compound can be synthesized as shown in Scheme Al. Scheme Al:
Figure GB2552041A_D0213
ΌΗ
1. Tf2O
2. NaNO2/ H2O
3. Tf2O
Figure GB2552041A_D0214
N3
0H ii
1. DMTrCl
2. H2
Figure GB2552041A_D0215
'NHCbz
4. NaN3
5. TBAF
3. CbzCI
4. CEM
HO
DMTrO γ
Figure GB2552041A_D0216
O
Figure GB2552041A_D0217
o
I
1. TFA/pyr
2. deprotect
3. deprotect
O
Figure GB2552041A_D0218
'NHCbz
OPO2H iv
1. hydrogenate
2. deprotect
Figure GB2552041A_D0219
deprotect
Figure GB2552041A_D0220
Figure GB2552041A_D0221
O
Figure GB2552041A_D0222
O [0371] Synthesis of the C-2’ amino cyclic dinucleotide (viii) can be accomplished using a multistep synthesis as outlined in scheme Al above and described below in EXAMPLE 3.
Synthesis of ATAC Compounds [0372] An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC1 - ATAC4, can be synthesized as shown in Scheme BI.
Scheme B1:
-1901.
u u
AISC)
O O
R. Zo.
O— O /O' ' n
Figure GB2552041A_D0223
2. DIC/ ROH
R = NHS, pentafluorophenyl ISC: immune-stimulatory compound [0373] A PEGylated carboxylic acid (i) that has been activated for amide bond formation can be reacted with an appropriately substituted amine containing immune-stimulatory compound to afford an intermediate amide. Formation of an activated ester (ii) can be achieved by reaction the intermediate amide-containing carboxylic using a reagent such as N-hydroxysuccinimide or pentafluorophenol in the presence of a coupling agent such as diisopropylcarbodiimide (DIC) to provide compounds (ii).
[0374] An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC5 - ATAC10, can be synthesized as shown in Scheme B2.
Scheme B2:
r3ox
Figure GB2552041A_D0224
i
Figure GB2552041A_D0225
no2 r3o·
O Ri ,, O H s I H
Figure GB2552041A_D0226
ISC ii
Figure GB2552041A_D0227
R4 = NHS, Perfluorofenyl
ISC: immune-stimulatory compound [0375] An activated carbonate such as (i) can be reacted with an appropriately substituted amine containing immune-stimulatory compound to afford carbamates (ii) which can be deprotected using standard methods based on the nature of the R3 ester group. The resulting carboxylic acid (iii) can
-191then by coupled with an activating agent such as N-hydroxysuccinimide or pentafluorophenol to provide compounds (iv).
[0376] An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC 11 - ATAC21, can be synthesized as shown in Scheme B3.
Scheme B3:
Figure GB2552041A_D0228
i-a; X = NHS jj i-b; X = H
ISC; immune-stimulatory compound [0377] An activated carboxylic ester such as (i-a) can be reacted with an appropriately substituted amine containing immune-stimulatory compound to afford amides (ii). Alternatively, carboxylic acids of type (i-b) can be coupled to an appropriately substituted amine containing immunestimulatory compound in the presence of an amide bond forming agent such as dicyclohexycarbodiimde (DCC) to provide the desired ATAC compounds.
[0378] An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC22 - ATAC31, can be synthesized as shown in Scheme B4.
Scheme B4:
o
Figure GB2552041A_D0229
i
Figure GB2552041A_D0230
no2
O ,0
Figure GB2552041A_D0231
Figure GB2552041A_D0232
H ii
ISC: immune-stimulatory compound [0379] An activated carbonate such as (i) can be reacted with an appropriately substituted amine containing immune-stimulatory compound to afford carbamates (ii) as the target ATAC compounds.
-192[0380] An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC32 - ATAC34, can be synthesized as shown in Scheme B5. Scheme B5:
Figure GB2552041A_D0233
ISC: immune-stimulatory compound [0381] An activated carboxylic acid such as (i-a, i-b, i-c) can be reacted with an appropriately substituted amine containing immune-stimulatory compound to afford amides (ii-a, ii-b, ii-c) as the target ATAC compounds.
[0382] These antibody construct immune-stimulatory conjugates can be made by various methods. It is understood that one skilled in the art may be able to make these compounds by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described herein by using the appropriate starting materials and modifying the synthetic route as needed. Starting materials and reagents can be obtained from commercial vendors or synthesized according to sources known to those skilled in the art or prepared as described herein.
Pharmaceutical Formulations [0383] The compositions and methods described herein can be considered useful as pharmaceutical compositions for administration to a subject in need thereof. Pharmaceutical compositions can comprise at least the compositions described herein and one or more pharmaceutically acceptable carriers, diluents, excipients, stabilizers, dispersing agents, suspending agents, and/or thickening
-193agents. The composition can comprise the conjugate having an antibody construct and an agonist. The composition can comprise the conjugate having an antibody construct, a targeting binding domain, and an agonist. The composition can comprise any conjugate described herein. Often, the antibody construct is an anti-CD40 antibody. A conjugate can comprise an anti-CD40 antibody and a PAMP molecule. A conjugate can comprise an anti-CD40 antibody and a DAMP molecule. A pharmaceutical composition can further comprise buffers, antibiotics, steroids, carbohydrates, drugs (e.g., chemotherapy drugs), radiation, polypeptides, chelators, adjuvants and/or preservatives.
[0384] Pharmaceutical compositions can be formulated using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a composition as described herein can be manufactured, for example, by lyophilizing the conjugate, mixing, dissolving, emulsifying, encapsulating or entrapping the conjugate. The pharmaceutical compositions can also include the compositions described herein in a free-base form or pharmaceutically-acceptable salt form.
[0385] Methods for formulation of the conjugates described herein can include formulating any of the conjugates described herein with one or more inert, pharmaceutically-acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions can include, for example, powders, tablets, dispersible granules and capsules, and in some aspects, the solid compositions further contain nontoxic, auxiliary substances, for example wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives. Alternatively, the compositions described herein can be lyophilized or in powder form for re-constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use [0386] Pharmaceutical compositions of the conjugates described herein can comprise at least an active ingredient. The active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug-delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
[0387] Pharmaceutical compositions as described herein often further can comprise more than one active compound as necessary for the particular indication being treated. The active compounds can have complementary activities that do not adversely affect each other. For example, the composition can comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-194hormonal agent, anti-angiogenic agent, and/or cardioprotectant. Such molecules can be present in combination in amounts that are effective for the purpose intended.
[0388] The compositions and formulations can be sterilized. Sterilization can be accomplished by filtration through sterile filtration.
[0389] The compositions described herein can be formulated for administration as an injection. Nonlimiting examples of formulations for injection can include a sterile suspension, solution or emulsion in oily or aqueous vehicles. Suitable oily vehicles can include, but are not limited to, lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. The suspension can also contain suitable stabilizers. Injections can be formulated for bolus injection or continuous infusion. Alternatively, the compositions described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0390] For parenteral administration the conjugates can be formulated in a unit dosage injectable form (e g., use letter solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles can be inherently nontoxic, and non-therapeutic. A vehicles can be water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used. Liposomes can be used as carriers. The vehicle can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability (e.g., buffers and preservatives).
[0391] Sustained-release preparations can be also be prepared. Examples of sustained-release preparations can include semipermeable matrices of solid hydrophobic polymers that can contain the antibody, and these matrices can be in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices can include polyesters, hydrogels (e.g., poly(2-hydroxyethylmethacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and γ ethyl-Lglutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPO™ (i.e., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-( - )-3-hydroxybutyric acid.
[0392] Pharmaceutical formulations of the compositions described herein can be prepared for storage by mixing a conjugate with a pharmaceutically acceptable carrier, excipient, and/or a stabilizer. This formulation can be a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, and/or stabilizers can be nontoxic to recipients at the dosages and concentrations used. Acceptable carriers, excipients, and/or stabilizers can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives,
-195polypeptides; proteins, such as serum albumin or gelatin; hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes; and/or non-ionic surfactants or polyethylene glycol.
Therapeutic Applications [0393] The compositions and methods of the present disclosure can be useful for a plurality of different subjects including, but are not limited to, a mammal, human, non-human mammal, a domesticated animal (e.g., laboratory animals, household pets, or livestock), non-domesticated animal (e.g., wildlife), dog, cat, rodent, mouse, hamster, cow, bird, chicken, fish, pig, horse, goat, sheep, rabbit, and any combination thereof.
[0394] The compositions and methods described herein can be useful as a therapeutic, for example a treatment that can be administered to a subject in need thereof. A therapeutic effect of the present disclosure can be obtained in a subject by reduction, suppression, remission, or eradication of a disease state, including, but not limited to, a symptom thereof. A therapeutic effect in a subject having a disease or condition, or pre-disposed to have or is beginning to have the disease or condition, can be obtained by a reduction, a suppression, a prevention, a remission, or an eradication of the condition or disease, or pre-condition or pre-disease state.
[0395] In practicing the methods described herein, therapeutically-effective amounts of the compositions described herein can be administered to a subject in need thereof, often for treating and/or preventing a condition or progression thereof. A pharmaceutical composition can affect the physiology of the subject, such as the immune system, inflammatory response, or other physiologic affect. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors. [0396] Treat and/or treating can refer to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. Treat can be used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and can contemplate a range of results directed to that end, including but not restricted to prevention of the condition entirely.
[0397] Prevent, preventing and the like can refer to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other
-196form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
[0398] A therapeutically effective amount can be the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental nonbeneficial event to the individual to whom the composition is administered. A therapeutically effective dose can be a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. An exact dose can depend on the purpose of the treatment, and can be ascertainable by one skilled in the art using known techniques.
[0399] The conjugates described herein that can be used in therapy can be formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, the condition of the individual patient, the site of delivery of the composition, the method of administration and other factors known to practitioners. The conjugates described herein can be prepared according to the description of preparation described herein.
[0400] Pharmaceutical compositions can be considered useful with the compositions and methods described herein can be administered to a subject in need thereof using a technique known to one of ordinary skill in the art which can be suitable as a therapy for the disease or condition affecting the subject. One of ordinary skill in the art would understand that the amount, duration and frequency of administration of a pharmaceutical composition described herein to a subject in need thereof depends on several factors including, for example but not limited to, the health of the subject, the specific disease or condition of the patient, the grade or level of a specific disease or condition of the patient, the additional therapeutics the subject is being or has been administered, and the like.
[0401] The methods and compositions described herein can be for administration to a subject in need thereof. Often, administration of the compositions described herein can include routes of administration, non-limiting examples of administration routes include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrasternal, intratumoral, or intraperitoneally. Additionally, a pharmaceutical composition can be administered to a subject by additional routes of administration, for example, by inhalation, oral, dermal, intranasal, or intrathecal administration. [0402] Compositions of the present disclosure can be administered to a subject in need thereof in a first administration, and in one or more additional administrations. The one or more additional administrations can be administered to the subject in need thereof minutes, hours, days, weeks or months following the first administration. Any one of the additional administrations can be
-197administered to the subject in need thereof less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days or less than 1 day after the first administration. The one or more administrations can occur more than once per day, more than once per week or more than once per month.
Diseases, Conditions and the Like [0403] The compositions and methods provided herein can be useful for the treatment of a plurality of diseases, conditions, preventing a disease or a condition in a subject or other therapeutic applications for subjects in need thereof. Often the compositions and methods provided herein can be useful for treatment of hyperplastic conditions, including but not limited to, neoplasms, cancers, tumors and the like. A condition, such as a cancer, can be associated with expression of a molecule on the cancer cells. Often, the molecule expressed by the cancer cells can comprise an extracellular portion capable of recognition by the antibody portion of the conjugate. A molecule expressed by the cancer cells can be a tumor antigen. An antibody portion of the conjugate can recognize a tumor antigen. A tumor antigen can include CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, pl85HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC1, MUC15, MSLN, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, or Fosrelated antigen 1.
[0404] As described herein, an antigen binding domain portion of the conjugate, can be configured to recognize a molecule expressed by a cancer cell, such as for example, a disease antigen, tumor antigen or a cancer antigen. Often such antigens are known to those of ordinary skill in the art, or
-198newly found to be associated with such a condition, to be commonly associated with, and/or, specific to, such conditions. For example, a disease antigen, tumor antigen or a cancer antigen is, but is not limited to, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folatebinding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Ley, CA-125, CA19-9, epidermal growth factor, pl85HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin BI, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, R0R2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, or Fos-related antigen 1. Additionally, such tumor antigens can be derived from the following specific conditions and/or families of conditions, including but not limited to, cancers such as brain cancers, skin cancers, lymphomas, sarcomas, lung cancer, liver cancer, leukemias, uterine cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, hemangiosarcomas, bone cancers, blood cancers, testicular cancer, prostate cancer, stomach cancer, intestinal cancers, pancreatic cancer, and other types of cancers as well as pre-cancerous conditions such as hyperplasia or the like.
[0405] Non-limiting examples of cancers can include Acute lymphoblastic leukemia (ALL); Acute myeloid leukemia; Adrenocortical carcinoma; Astrocytoma, childhood cerebellar or cerebral; Basalcell carcinoma; Bladder cancer; Bone tumor, osteosarcoma/malignant fibrous histiocytoma; Brain cancer; Brain tumors, such as, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, visual pathway and hypothalamic glioma; Brainstem glioma; Breast cancer; Bronchial adenomas/carcinoids; Burkitt's lymphoma; Cerebellar astrocytoma; Cervical cancer; Cholangiocarcinoma; Chondrosarcoma; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon cancer; Cutaneous T-cell lymphoma; Endometrial cancer; Ependymoma; Esophageal cancer; Eye cancers, such as, intraocular melanoma and retinoblastoma; Gallbladder cancer; Glioma; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Islet cell
-199carcinoma (endocrine pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal cancer; Leukaemia, such as, acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous and, hairy cell; Lip and oral cavity cancer; Liposarcoma; Lung cancer, such as, nonsmall cell and small cell; Lymphoma, such as, AIDS-related, Burkitt; Lymphoma, cutaneous T-Cell, Hodgkin and Non-Hodgkin, Macroglobulinemia, Malignant fibrous histiocytoma of bone/osteosarcoma; Melanoma; Merkel cell cancer; Mesothelioma; Multiple myeloma/plasma cell neoplasm; Mycosis fungoides; Myelodysplastic syndromes; Myelodysplastic/myeloproliferative diseases; Myeloproliferative disorders, chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Oligodendroglioma; Oropharyngeal cancer; Osteosarcoma/malignant fibrous histiocytoma of bone; Ovarian cancer; Pancreatic cancer; Parathyroid cancer; Pharyngeal cancer; Pheochromocytoma; Pituitary adenoma; Plasma cell neoplasia; Pleuropulmonary blastoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Rhabdomyosarcoma; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (non-melanoma); Skin carcinoma; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma; Squamous neck cancer with occult primary, metastatic;
Stomach cancer; Testicular cancer; Throat cancer; Thymoma and thymic carcinoma; Thymoma,; Thyroid cancer; Thyroid cancer, childhood; Uterine cancer; Vaginal cancer; Waldenstrom macroglobulinemia; Wilms tumor and any combination thereof.
EXAMPLE 1
Fc Receptor Binding to anti-CD40 Antibodies [0406] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and two light chain from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody. An anti-CD40 antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains from a SBT040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-CD40 antibody is comprised two SBT-040-G1DE heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is comprised of two SBT-040G1 AAA heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT040-AAA antibody.
[0407] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and SBT-040AAA antibody are produced by standard methods for producing antibodies. These antibodies are purified, and each antibody’s affinity for soluble glycosylated ectodomains from all human Tcy
-200receptors (FcyRs) is measured. These affinities are measured by experiments using surface plasmon resonance. In these experiments, biotinylated soluble glycosylated FcyR ectodomains from all human FcyRs are immobilized on a streptavidin-coated surface. The ability of each antibody to bind to soluble glycosylated FcyR ectodomains from all human FcyRs is then measured by surface plasmon resonance using a Biacore instrument. The data from this experiment shows that the Fc domain of a SBT-040-WT antibody, the Fc domain of a SBT-040-VFPFF antibody, the Fc domain of a SBT-040-DE antibody, and the Fc domain of a SBT-040-AAA antibody are each bound to soluble glycosylated FcyR ectodomains from all human FcyRs. Therefore, the surface plasmon resonance experiments show that the Fc domain of the SBT-040-G1WT antibody and variants of the Fc domain of a SBT-040-G1WT antibody (i.e., the Fc domain of a SBT-040-G1VFPFF antibody, the Fc domain of a SBT-040-DE antibody and the Fc domain of a SBT-040-AAA antibody) are each bound to all human FcyRs. The affinity of each antibody for each human FcyRs is also shown by these experiments.
EXAMPLE 2
Synthesis of Linkers with Immune-Stimulatory Compounds [0408] A linker is linked with an immune-stimulatory compound. A linker linked to an immunestimulatory compound is formed to make a linker-immune stimulatory compound conjugate (ATAC). Subsequently, an ATAC is conjugated to an antibody, in which the ATAC is any one of ATAC1 - ATAC34 or ATAC 43 (each of which is described in the below EXAMPLES).
[0409] A linker is linked with an antibody, in which the linker is a pegylated linker, a valine-alanine linker, a valine-citrulline linker, or an N-Maleimidomethylcyclohexane-l-carboxylate (MCC) linker. Subsequently, an immune-stimulatory compound is conjugated to the linker linked with the antibody, in which the immune-stimulatory compound is a TLR ligand, a Nod-like receptor ligand, a RIG-Like receptor ligand, a CLR ligand, a CDS ligand, or an inflammasome inducer.
[0410] A linker is linked with an antibody, in which the linker is a pegylated linker, a valine-alanine linker, a valine-citrulline linker, or an N-Maleimidomethylcyclohexane-l-carboxylate (MCC) linker. Subsequently, an immune-stimulatory compound is conjugated to the linker linked with the antibody, in which the immune-stimulatory compound is gardiquimod or an analog of a cyclic dinucleotide.
EXAMPLE 3
-201Synthesis of (lR,6R,8R,9R,10S,15R,17R,18R)-9-Amino-8,17-bis(2-amino-6-oxo-l,9dihydropurin-9-yl)-3,12-dihydroxy-18-hydroxy-2.4.7.11.13.16-hexaoxa-3Z5.12Z5diphosphatricyclo [13.3.0.06,10]octadecane-3,12-dione (Compound 21) [0411] This example shows the synthesis of (lR,6R,8R,9R,10S,15R,17R,18R)-9-Amino-8,17-bis(2amino-6-oxo-l,9-dihydropurin-9-yl)-3,12-dihydroxy-18-hydroxy-2.4.7.11.13.16-hexaoxa-3k5.12Z5diphosphatricyclo [13.3.0.06,10]octadecane-3,12-dione (Compound 21).
o
Figure GB2552041A_D0234
o
Compound 21
Step A: Preparation of Int 2.13-1
Figure GB2552041A_D0235
1. TBSCI, Py, 25°C, 3h
2. isobutyric anhydride, 25°C, 16h
3. NH3H2O, 25°C, 1h
Figure GB2552041A_D0236
Int 2.13-1 [0412] Guanosine (200 g, 706.71 mmol, 1.00 equiv) was suspended in dry pyridine (4000 mL) under a nitrogen atmosphere, and TBSCI (572 g, 5.30 mol, 7.50 equiv) was added dropwise at 0°C. The reaction was stirred at ambient temperature for 3h then cooled to 0°C before adding isobutyric anhydride (167 g, 1.06 mol, 1.5 equiv) dropwise over 20 min. The solution was allowed to warm to room temperature and stirred for 16h. The reaction solution was cooled to 0°C and the reaction was quenched by the addition of water (500 mL). After stirring for 20 min at 0°C, 1000 mL of concentrated aqueous NH40H was added dropwise at 0°C. After stirring for an additional lh at room temperature, the resulting mixture was concentrated and the residue was dissolved in 3000 mL of water and washed with 1500 mL of EtOAc. The aqueous phase was concentrated to -1000 mL whereby the product precipitated from water. The product was filtered to afford 174 g ofN-[9[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2methylpropanamide (Int 2.13-1) as a white solid.
-202Step B: Preparation of Int 2.13-2
Figure GB2552041A_D0237
Int 2.13-1
Figure GB2552041A_D0238
Int 2.13-2 [0413] To a stirred suspension of Int 2.13-1 (200.0 g, 566.57 mmol, 1.00 equiv) in pyridine (3 L) under a nitrogen atmosphere was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (211 g, 623.23 mmol, 1.10 equiv). The resulting mixture was left to stir for 16 h at room temperature. The reaction was quenched with methanol (100 mL) and the mixture was concentrated under vacuum. The residue was dissolved in 3000 mL of dichloromethane, washed with 2 x 1500 mL of saturated sodium bicarbonate solution and 1500 mL of saturated sodium chloride solution respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum.
The crude product was applied onto a silica gel column with DCM/methanol (with 0.05% triethylamine) (50/1-20/1). This resulted in 278 g (75%) ofN-[9-[(2R,3R,4S,5R)-5-[[bis(4methoxyphenyl)(phenyl)methoxy]methyl]-3,4-dihydroxyoxolan-2-yl]-6-oxo-6,9-dihydro-lH-purin2-yl]-2-methylpropanamide (Int 2.13-2) as a light yellow solid.
Step C: Preparation of Int 2.13-3a and Int 2.13-3b
DMTrO
Z' Ti nh
TBSCI, Py, Im DMTrO H r
25°C,16h
OH OH
Int 2.13-2
Λ? «X
OH OTBS
Int 2.13-3a
NH °
n-^n*X
H
TBSO OH
Int 2.13-3b
Figure GB2552041A_D0239
[0414] Compound Int 2.13-2 (150 g, 229 mmol, 1.00 equiv) was dissolved in 1500 mL of pyridine under a nitrogen atmosphere. lH-imidazole (46.71 g, 687.02 mmol, 3.00 equiv) was added, followed by addition of TBS-C1 (51.6 g, 343.51 mmol, 1.50 equiv) in portions at 25 °C. The resulting solution was stirred for 16 h at 25 °C then concentrated and dissolved in 2000 mL of di chloromethane. The organic extract was washed with 2 x 1000 mL of saturated sodium bicarbonate solution and 1000 mL of saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was applied onto a silica
-203gel column with ethyl acetate/dichloromethane (1/50-1/1) and then purified by flash with the following conditions: silica gel column; ethyl acetate in dichloromethane with 0.05% triethylamine:15% up to 70% within 10 min and 70% maintained 10 min; This resulted in 62 g (35%) ofN-[9-[(2R,3R,4R,5R)-5-[[bis(4-methoxyphenyl)(phenyl)methoxy]methyl]-3-[(tertbutyldimethylsilyl)oxy]-4-hydroxyoxolan-2-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2methylpropanamide (Int 2.13-3a) as a yellow solid and 44 g (25%) of N-[9-[(2R,3R,4S,5R)-5[[bis(4-methoxyphenyl)(phenyl)methoxy]methyl]-4-[(tert-butyldimethylsilyl)oxy]-3-hydroxyoxolan2-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2-methylpropanamide (Int 2.13-3b).
Step D: Preparation of Int 2.13-4 o
Figure GB2552041A_D0240
Int2.13-3a Int 2.13-4 [0415] Int 2.13-3a (28 g, 36.41 mmol, 1.00 equiv) was dissolved in 280 mL of dichloromethane under a nitrogen atmosphere. lH-imidazole-4,5-dicarbonitrile (12.9 g, 109.23 mmol, 3.00 equiv) and 3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (43.84 g, 145.64 mmol, 4.00 equiv) were added in order. The resulting solution was stirred for 1 h at 25°C and the resulting solution was diluted with 500 mL of dichloromethane and washed with 4 x 400 mL of saturated sodium bicarbonate solution and 1x400 mL of saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by flash chromatography (Cl8 silica gel; mobile phase, acetonitrile in water gradient :40% up to 100% within 8 min and 100% maintained 10 min) to afford 25 g of Int 2.13-4 as a white solid.
Step E: Preparation of Int 2.13-5
-204ο
Figure GB2552041A_D0241
ο
Int 2.13-1
Int 2.13-5 [0416] To a solution of Int 2.13-1 (190 g, 538.24 mmol) in 3000 mL of pyridine was added 1,1,3,3tetraisopropyl-l,3-dichlorodisiloxane (152.6 g, 484.42 mmol, 0.9 equiv) dropwise at 0°C. The resulting solution was stirred for 16 h at 25°C. The reaction was quenched by the addition of 30 mL of methanol and the resulting solution was concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (50/1-30/1) to afford 189 g ofN-[9[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetrakis(propan-2-yl)-hexahydro-2H-furo[3,2f][l,3,5,2,4]trioxadisilocin-8-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2-methylpropanamide (Int 2.135) as a white solid.
Step F: Preparation of Int 2.13-6
Figure GB2552041A_D0242
Int 2.13-5 Int 2.13-6 [0417] Into a 250-mL 3-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed a solution of 195 g (327.73 mmol) of N-[9-[(6aR,8R,9R,9aS)-9-hydroxy2,2,4,4-tetrakis(propan-2-yl)-hexahydro-2H-furo[3,2-f][l,3,5,2,4]trioxadisilocin-8-yl]-6-oxo-6,9dihydro-lH-purin-2-yl]-2-methylpropanamide (Int 2.13-5) in 4000 mL of dichloromethane, 129.5 g (5.00 equiv) of pyridine and 4-dimethylaminopyridine (20 g, 163.86 mmol, 0.50 equiv). The solution was cooled to 0°C and treated with 184.8 g (655.5 mmol, 2.0 equiv) triflic anhydride dropwise. The resulting solution was stirred for 2 h at 0 °C then quenched by the addition of 4000 mL of water/ice. The resulting solution was extracted with 3 x 4000 mL of di chloromethane and the organic layers were combined. The organic extracts were washed with 2 x 4000 mL of water/ice and 1 x 4000 mL
-205of saturated sodium chloride solution, respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to provide 213 g (crude) of (6aR,8R,9R,9aR)-8-[2-(2methylpropanamido)-6-oxo-6,9-dihydro-lH-purin-9-yl]-2,2,4,4-tetrakis(propan-2-yl)-hexahydro2H-furo[3,2-f][l,3,5,2,4]trioxadisilocin-9-yl trifluoromethanesulfonate (Int 2.13-6) as a yellow solid.
Step G: Preparation of Int 2.13-7
Figure GB2552041A_D0243
Int 2.13-6 Int 2.13-7 [0418] Int 2.13-6 (213 g, crude) was dissolved in 2100 mL of DMF and treated with sodium nitrite (158.3 g, 2.29 mol, 7.00 equiv). After stirring forl6 h the solution was filtered and concentrated under vacuum. The resulting solution was diluted with 6000 mL of DCM and washed with 2 x 3000 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane/methanol (50/1-30/1) and then purified using the following conditions: C18 silica gel, 50% MeOH/water to 100% water over 10 min then 100% water for 10 min to provide 50 g (26% for 2 steps) of Int 2.13-7 as a white solid.
Step H: Preparation of Int 2.13-8
Figure GB2552041A_D0244
Int 2.13-7 Int 2.13-8 [0419] N-[9-[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetrakis(propan-2-yl)-hexahydro-2H-furo[3,2-206f][l,3,5,2,4]trioxadisilocin-8-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2-methylpropanamide (Int 2.137) (50 g, 84.03 mmol) and DMAP (30.8 g, 252.10 mmol, 3.00 equiv) were dissolved in 500 mL of DCM and the mixture was cooled to 0°C. Triflic anhydride (30.8 g, 109.24 mmol, 1.30 equiv) was then added dropwise with stirring at 0°C. The resulting solution was stirred at this temperature for 1 h. The reaction was then quenched by the addition of 500 mL of ice/water then extracted with 3 x 500 mL of dichloromethane and the organic layers were combined. The organic layer was washed with 2 x 100 mL of saturated sodium chloride solution. The solution was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product (Int 2.13-8) (55 g) was thus isolated and used directly in the next step.
Step I: Preparation of Int 2.13-9
Figure GB2552041A_D0245
Int 2.13-8 Int 2.13-9 [0420] Int 2.13-8 (55g, crude) was dissolved in N,N-dimethylformamide (500 mL) then treated with sodium azide (27.8 g, 427.73 mmol, 5.1 equiv). The resulting solution was stirred for 16 h at room temperature. The resulting mixture was filtered and concentrated under vacuum. The residue was applied onto a silica gel column with dichloromethane/methanol (20/1) to afford 15.0 g of Int 2.13-9 as a yellow solid.
Step J: Preparation of Int 2.13-10
Figure GB2552041A_D0246
-207[0421] Int 2.13-9 (23 g, 1.00 equiv) in THF (230mL) was treated with tetrabutylammonium fluoride (37 mL, 1.0 equiv). The resulting solution was stirred for 10 min at room temperature then concentrated under vacuum. The residue was applied directly to a silica gel column with dichloromethane/methanol (100/1-20/1). This resulted in 12.4 g (88%) ofN-[9-[(2R,3R,4S,5R)-3azido-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2methylpropanamide (Int 2.13-10) as a white solid.
Step K: Preparation of Int 2.13-11
Figure GB2552041A_D0247
lnt2·13-10 Int 2.13-11 [0422] Into a 50-mL round-bottom flask, was placed a solution of 11 g of Int 2.13-10 in pyridine (60 mL). 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (14.75 g, 1.50 equiv) was added and the resulting solution was stirred for 2 h at room temperature. The reaction was quenched by the addition of 20 mL of methanol and the resulting mixture was concentrated under vacuum. The residue was dissolved in 500 mL of dichloromethane, washed with 2 x 250 mL of saturated sodium bicarbonate solution and 1 x 250 mL of saturated sodium chloride solution respectively. The organic phase was dried over sodium sulfate, filtered and concentrated under vacuum. The residue was chromatographed with dichloromethane/methanol with 0.05% triethylamine (100/1-60/1) to afford 17.8 g of Int 2.13-11 as a white solid.
Step L: Preparation of Int 2.13-12
Figure GB2552041A_D0248
OH N3 MeOH OH NH2
25°C, 1h
Int 2.13-11 Int 2.13-12
-208[0423] Into a 50-mL round-bottom flask was placed a solution of 10 g of Int 2.13-11 in methanol (150 mL) and 10% anhydrous palladium on carbon (2g, w/w). The resulting mixture was stirred under an atmosphere of hydrogen for lh at room temperature. The mixture was filtered through
Celite and concentrated to provide 8.7 g of Int 2.13-12 as a yellow solid which was used directly without further purification.
Step M: Preparation of Int 2.13-13
DMTrO
Figure GB2552041A_D0249
Int 2.13-12
CbzCI, NaHCO3
THF, H2O 25°C, 20min
DMTrO
Figure GB2552041A_D0250
Int 2.13-13 [0424] To a solution of 16.8 g of N-[9-[(2R,3R,4S,5R)-3-amino-5-[[bis(4-methoxyphenyl)(phenyl)methoxy]methyl]-4-hydroxyoxolan-2-yl]-6-oxo-6,9-dihydro-lH-purin-2-yl]-2-methylpropanamide (Int 2.13-12) in 210 mL of THF/water (4/1) was added sodium bicarbonate (6.46g, 3.00 equiv) followed by the addition of Cbz-Cl (5.67g, 1.30 equiv). The resulting solution was stirred for 20 min at room temperature. The reaction was diluted with saturated sodium carbonate solution (100 mL) and extracted with 3 x 200 mL of dichloromethane. The organic phase was dried over sodium sulfate, filtered and concentrated under vacuum. The residue was chromatographed with dichloromethane/methanol with 0.05% triethylamine (100/1) and then crystallized from dichloromethane (40 mL) to afford 14.8g (86% over two steps) of Int 2.13-13 as a white solid.
Step N: Preparation of Int 2.13-14 o
Figure GB2552041A_D0251
0H fiHCbz
O
Figure GB2552041A_D0252
Int 2.13-14
Int 2.13-13 [0425] Int 2.13-13 (14.8 g, 18.78 mmol) was dissolved in 150 mL dichloromethane under a nitrogen
-209atmosphere, 3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (22.6 g, 75.12 mmol, 4.00 equiv) and lH-imidazole-4,5-dicarbonitrile (6.64 g, 56.34 mmol 3.00 equiv) were added in order.
The resulting solution was stirred for 1 h at 25°C and diluted with 400 mL of dichloromethane. The solution was washed with 1 x 500 mL of saturated sodium bicarbonate solution and 1 x 500mL of saturated sodium chloride solution respectively. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by Flash with the following conditions: Column, C18 silica gel; mobile phase, acetonitrile in water: 30% up to 80% within 8 min and 100% maintained 10 min to provide 14.8 g (75%) of N-[9-[(2R,3R,4R,5R)-5[[bis(4-methoxyphenyl)(phenyl)methoxy]methyl]-3-([[bis(propan-2-yl)amino](2cyanoethoxy)phosphanyl]oxy)-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]-6-oxo-6,9-dihydro-lHpurin-2-yl]-2-methylpropanamide (Int 2.13-14) as a white solid.
Step O: Preparation of Int 2.13-15
Figure GB2552041A_D0253
Int 2.13-14
Figure GB2552041A_D0254
Int 2.13-15 [0426] To a solution of Int 2.13-14 (7.2 g, 7 .20 mmol) in acetonitrile (600 mL) and water (260 mg) was added pyridinium triflate (1.66g, 8.38 mmol, 1.20 equiv). The resulting solution was stirred for 10 min at room temperature to provide a solution of Int 2.13-15 which was used directly in the next step.
Step P: Preparation of Int 2.13-16
Figure GB2552041A_D0255
Ι nt 2.13-16
Int 2.13-15
-2 ΙΟ[0427] The solution containing Int 2.13-15 was treated with tert-butyl amine (36 mL) for 30 min at room temperature. The mixture was then concentrated under vacuum to afford 7.5 g of Int 2.13-16 as a foam which was used directly at next step without further purification.
Step Q: Preparation of Int 2.13-17
Figure GB2552041A_D0256
O=FJ~H
O’ tBuNH3 +
CI2CHCO2H, PCM
25°C, 10min
Figure GB2552041A_D0257
O—P_H
OH
Int 2.13-17
Int 2.13-16 [0428] Int 2.13-16 was dissolved in 75 mL of dichloromethane and the solution was treated with 91.5 mL of 6% dichloroacetic acid in dichloromethane. Triethylsilane (150 ml) was added after 10 minutes followed by pyridine (10 mL). The mixture was concentrated and the residue was dissolved in 50 mL of dry acetonitrile and concentrated. This process was repeated twice. The residue was finally dissolved in 20 mL of acetonitrile and used directly at next step.
Figure GB2552041A_D0258
Figure GB2552041A_D0259
[0429] To the above solution containing 3.0 g of Int 2.13-17 and 3.0 g of 4A MS in 20 mL of MeCN was added a solution of Int 2.13-4 (18 g, 18.56 mmol, 2.5 equiv) in 30 ml of dry acetonitrile. The reaction solution was stirred for 5 min at 25°C and used directly in the next step.
-211StepS: Preparation of Int 2.13-19
Figure GB2552041A_D0260
[0430] To above reaction solution (Int 2.13-18) was added 8 mL of tert-butyl hydroperoxide (5.5 M in decane). The resulting solution was stirred for 30 min at 25°C then diluted with 250 mL of ethyl acetate. The resulting solution was washed with 2 x 300 mL of water and 1 x 300 mL of saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to afford 21 g of Int 2.13-19 which was used directly in the next step.
Step T: Preparation of Int 2.13-20
DMTrO </Nl XH
Ί Ο ΐ N N vy H
Figure GB2552041A_D0261
NCX___ Λ xo
OTBS.N-f NH
p. Λ J bl N N-\ ο h y ,o NHCbz
HO H °x/~
Int 2.13-19
Figure GB2552041A_D0262
[0431] Int 2.13-19 (21 g, crude) was dissolved in methylene chloride (250 mL) before adding 210 mL dichloroacetic acid (6% in methylene chloride) to the solution. After stirring for 10 min, 420 mL of triethylsilane was added while stirring at room temperature. 50 mL of pyridine was then added and the mixture was concentrated under vacuum. The resulting residue was purified by flash column chromatography (C18 gel column, mobile phase, acetonitrile/water with 0.04% ammonium
-212bicarbonate : 20% up to 80% within 10 min, then 100% for 5 min). This resulted in 2.5 g (30% overall for 6 steps) of Int 2.13-20 as a white solid.
StepU: Preparation of Int 2.13-21
Figure GB2552041A_D0263
lnt 2 -13-2θ Int 2.13-21 [0432] Compound 2-chloro-5,5-dimethyl-l,3,2-dioxaphosphorinane-2-oxide (1.9 g, 10.39 mmol, 4.7 eq) was added to the solution of Int 2.13-20 (2.5 g, 2.21 mmol) in 50 mL of pyridine and the reaction solution was stirred for 15 min at 25°C. The reaction was quenched by water (600 uL) followed by iodine (0.84 g, 3.31 mmol, 1.5 equiv). After stirring for 20 min, 12.5 mL saturated sodium thiosulfate solution was added. The mixture was concentrated to a foam to provide 2.5 g of Int 2.13-21 which was used directly in the next step.
StepV: Preparation of Int 2.13-22
Figure GB2552041A_D0264
Int 2.13-21 lnt 2.13-22 [0433] To a solution of 2.5 g crude Int 2.13-21 in acetonitrile (12.5 mL) was added tert-butylamine (22.5 mL). After stirring for 10 min at 25°C, the reaction solution was concentrated to a yellow foam and the crude product was purified by flash chromatography (Cl8 gel column, mobile phase, acetonitrile/water with 0.04% ammonium bicarbonate, gradient: 10% up to 50% within 15 min, 100% for 5 min) to provide pure 1.2 g (50% over 2 steps) Int 2.13-22.
-213Step W: Preparation of Int 2.13-23
Figure GB2552041A_D0265
[0434] A 50-mL round-bottom flask was purged with argon then charged with a solution of Int
2.13- 22 (500 mg, 0.46 mmol) in 25 mL of methanol. 10% anhydrous palladium carbon (250 mg, w/w) was then added and hydrogen was bubbled through the solution. The reaction mixture was stirred for 1 h at 25°C then filtered through Celite and concentrated to provide 380 mg (87%) of Int
2.13- 23 as a white solid.
Step X: Preparation of Int 2.13-24
Figure GB2552041A_D0266
o
I nt 2.13-23 Int 2.13-24 [0435] Into a 100-mL round-bottom flask was placed Int 2.13-23 (380 mg, 0.40 mmol) and 25 mL of methylamine (33% in anhydrous ethanol). The resulting solution was stirred for 16 h at 25°C then concentrated to provide Int 2.13-24 as a white foam which used directly in the next step.
Step Ύ: Preparation of Compound 21
-214H2Nx^N HN
Figure GB2552041A_D0267
Figure GB2552041A_D0268
°'P0 OH O <Λη N NH;
T Tz> hn^An o
O °4'o' o
nh2
1.82 NH4+
0.18
NH
Int 2.13-24
Compound 21 [0436] The above crude Int 2.13-24 was azeotroped with pyridine/triethylamine (9mL/3mL) three times then dissolved in 0.8 mL pyridine in a 100 mL round-bottom flask. To this solution at 55 °C was added 6 mL triethylamine and 4 mL triethylamine trihydrofluride simultaneously. After stirring lh, the bath was removed and 60 mL anhydrous acetone was added immediately. The mixture was stirred for 20 min and the white solid was collected by filtration. The precipitate was washed with 5 mL of anhydrous acetone. The product was purified by preparative flash chromatography (AQ-C18 silica gel; mobile phase = acetonitrile /water with 0.04% ammonium bicarbonate; gradient 1% to 20% over 20 min, UV detector @ 210 nm). The resulting solution was lyophilized to provide 100 mg (36% for 2 steps) of Compound 21 as a white solid. LC-MS- SVT-001-1-24: (ESI, m/z)·. 690 [M+H]+ ΧΕ[ NMR (D2O): δ 7.86 (s, 1H); 7.83 (s, 1H); 5.80-5.92 (m, 2H); 4.96-5.09 (m, 1H); 4.84-4.88 (m, 1H); 4.69-4.83 (m, 1H); 4.32-4.39 (m, 1H); 4.11-4.39 (m, 4H); 3.91-4.02 (m, 2H); [0.18 equiv TEA : 2.98-3.12 (q, J=6.6, 1H); 1.05-1.24(m, J=6.6, 1.6H)]. 31P NMR (D2O) δ-E28,-E39.
EXAMPLE 4
Synthesis of ATAC1 and ATAC2 [0437] This example shows the synthesis of Pentafluorophenyl 25-(2-amino-3-pentylquinolin-5-yl)19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC1) and Perfluorophenyl 3-((4-amino-l(2-hydroxy-2-methylpropyl)- lH-imidazo[4,5-c]quinolin-2-yl)methyl)-4-oxo-7,10,13,16,19pentaoxa-3-azadocosan-22-oate (ATAC2).
Figure GB2552041A_D0269
-215Step A: Preparation of Int AT AC 1-1
Figure GB2552041A_D0270
Figure GB2552041A_D0271
[0438] To a 0°C solution containing 271 mg (0.90 mmol) of 5-(5-aminopentyl)-3-pentylquinolin-2amine in 4 mL of DCM was added 435 mg (1.00 mmol) of the NHS ester in 1 mL of DCM dropwise over 15 minutes. The reaction mixture was allowed to warm to ambient temperature over 19h before it was concentrated and purified by reverse phase chromatography. Pure fractions were lyophilized and dissolved in 3 mL of methanol then treated with 1 mL of 4N HCI in dioxane. The solution was stirred for lh then concentrated to afford the desired compound as an HCI salt which was used directly in the next step.
Step B: Preparation of ATAC1
Figure GB2552041A_D0272
[0439] To a stirred solution of 25-(2-amino-3-pentylquinolin-5-yl)-19-oxo-4,7,10,13,16-pentaoxa-20 azapentacosanoic acid hydrochloride (130 mg, 0.198 mmol) and pentafluorophenol (146 mg, 0.792
-216mmol) in DMF (2.5 ml) at room temperature was added Ν,Ν'-Diisopropylcarbodiimide (0.186 ml, 1.189 mmol) dropwise. The reaction was stirred at room temperature for 18h then concentrated. The crude product was added to a lOOg Cl8 gold reverse phase column and was eluted with water/acetonitrile (0.1% TFA) 10-100%. The fractions were combined and concentrated then freeze dried to give perfluorophenyl 25-(2-amino-3-pentylquinolin-5-yl)-19-oxo-4,7,10,13,16-pentaoxa-20azapentacosanoate-2,2,2-trifluoroacetate (110 mg, 61.7% yield) as a clear gum. Ή NMR (DMSOd6) δ 13.7 (s, 1H), 8.37-8.35 (m, 3H), 7.78 (t, J=5 5Hz, 1H), 7.63 (t, J=7.5Hz, 1H), 7.53 (d, J=8.5Hz, 1H), 7.31 (d, J=7.0 Hz, 1H), 3.58 (t, J=6.0Hz, 2H), 3.63-3.43 (m, 20H), 3.04-2.96 (m, 6H), 2.73 (t, J=7.5Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J=7.5Hz, 3H). LCMS [M+H] = 786.3.
[0440] The following compound in TABLE 2 can be prepared using a method similar to that described above for ATAC1.
TABLE 2
Compound Structure IUPAC M+l
ATAC2 h2n F F N V fAXf ° ° -Y t HO \ Perfluorophenyl 3-((4-amino- l-(2-hydroxy-2- methylpropyl)- 1H- imidazo [4,5 -c]quinolin-2- yl)methyl)-4-oxo- 7,10,13,16,19-pentaoxa-3 - azadocosan-22-oate 800
EXAMPLE 5
Synthesis of ATAC3 and ATAC4 [0441] This example shows the synthesis of pentafluorophenyl 25-(2-amino-3-pentylquinolin-5-yl)19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC3) and 2,5-Dioxopyrrolidin-l-yl 3-((4amino-l-(2-hydroxy-2-methylpropyl)-lH-imidazo-[4,5-c]quinolin-2-yl)methyl)-4-oxo7,10,13,16,19-pentaoxa-3 -azadocosan-22-oate (ATAC4).
-217HCI
Figure GB2552041A_D0273
Step A: Preparation of ATAC3 [0442] To a stirred solution of Int ATAC1-1 (185 mg, 0.282 mmol) and N-hydroxysuccinimide (130 mg, 1.128 mmol) inDMF (3 ml) was addedΝ,Ν'-diisopropylcarbodiimide (0.221 ml, 1.411 mmol) dropwise and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered and washed with acetonitrile and the filtrate was evaporated. The resulting residue was purified by silica gel Silica gel column chromatography (DCM / MeOH / HO Ac) to give 65 mg of the desired product as the acetic acid salt which was subsequently dissolved in 2 mL of DCM and treated with 2M HCI in diethyl ether. The solution was stirred for lh then concentrated and lyophilized to afford the desired compound as the HCI salt. 'H NMR (CDCfl) δ 15.2 (s, 1H),
8.15 (d, J=7.8Hz, 1H), 7.68 (d, J=7.9Hz, 1H), 7.55 (t, J=8.1Hz, 1H), 6.55 (bs, 1H), 3.98 (t, J=6.0Hz, 2H), 3.83-3.55 (m, 18H), 3.33-3.22 (m, 2H), 2.95-2.56 (m, 11H), 2.27 (t, J=7.5Hz, 2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J=7.5Hz, 3H). LCMS [M+H] = 717.3.
[0443] The following 2,5-Dioxopyrrolidin-l-yl 3-((4-amino-l-(2-hydroxy-2-methylpropyl)-lHimidazo-[4,5-c]quinolin-2-yl)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oate (ATAC4) compound can be prepared using a method similar to that described above for ATAC3.
Figure GB2552041A_D0274
ATAC4 ^NMRiCDCh) δ 14.9 (s, 1H), 8.88 (bs, 1H), 8.15 (d, 1H), 7.85 (d, 1H), 7.61 (t, 1H), 7.45 (t, 1H), 4.72 (s, 2H), 3.83 (m, 4H), 3.65-3.45 (m, 18H), 2.90-2.71 (m, 9H), 1.43 (t, J=7.0Hz, 3H), 1.33 (s, 6H). LCMS [M+H] = 731.
-218EXAMPLE 6
Synthesis of ATAC5, ATAC6 and ATAC7 [0444] This example shows the synthesis of 2,5-dioxopyrrolidin-l-yl 6-(((S)-l-(((S)-l-((4-((((5-(2amino-3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxopropan-2yl)amino)-3-methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC5), 2,5-dioxopyrrolidin-l-yl 7 (((S)-1 -((( S)-1 -((4-(((((4-amino-1 -(2-hydroxy-2-methylpropyl)- lH-imidazo[4,5-c]quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC6), and 2,5-dioxopyrrolidin-l-yl 7-(((S)-l-(((S)-l((4-(((((4-amino-l-(2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC7).
Figure GB2552041A_D0275
Step A: Preparation of Int ATAC5-1
Figure GB2552041A_D0276
Figure GB2552041A_D0277
Int ATAC5-1 [0445] A solution of 5-(5-aminopentyl)-3-pentylquinolin-2-amine (300 mg, 1.00 mmol) in 5 mL
DCM was stirred at room temperature under nitrogen for 10 min before tert-butyladipate-valinealanine-para-aminobenzyl-4-nitrophenylcarbonate (tBuAdip-va-PAB-OPNP, 656 mg, 1.00 mmol)
-219and DIPEA (0.26 ml, 1.5 mmol) in 3 mL of DCM were added and the mixture was stirred at room temperature overnight. The mixture was concentrated and purified by column chromatography.
Clean fractions were combined and evaporated and the residue was dissolved in 2 mL of DCM and treated with 2M HC1 in diethyl ether. The solution was stirred for lh then concentrated and lyophilized to afford the desired compound Int ATAC5-1 as the HC1 salt. MS m/z 761 (M)+.
Step B: Preparation of ATAC5
Figure GB2552041A_D0278
[0446] To a stirred solution of 6-(((S)-l-(((S)-l-((4-((((5-(2-amino-3-pentylquinolin-5yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 -oxobutan-2yl)amino)-6-oxohexanoic acid hydrochloride (221 mg, 0.282 mmol) and N-hydroxysuccinimide (130 mg, 1.12 mmol) in DML (3 ml) was added Ν,Ν'-diisopropylcarbodiimide (0.221 ml, 1.41 mmol) dropwise and the reaction mixture was stirred at room temperature for 5h. HPLC indicated some starting material remained so the reaction was stirred at ambient temperature overnight. The reaction mixture was filtered and washed with acetonitrile. The filtrate was evaporated and the residue was dissolved in DMSO and purified by reverse phase chromatography [water/acetonitrile (0.1% TFA)] from 10% followed by a gradient from 20 to 80%. Pure fractions were combined to give 2,5-dioxopyrrolidin-1 -yl 6-(((S)-1 -(((S)-1 -((4-((((5-(2-amino-3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 -oxobutan-2-yl)amino)6-oxohexanoate 2,2,2-trifluoroacetate (109 mg, 40 % yield) as a white solid. Ή NMR (DMSO-d6) δ 13.6 (s, 1H), 9.92 (s, 1H), 8.32 (d, J=7.5Hz, 1H), 7.84 (d, J=8.5Hz,lH), 7.61-7.55 (m, 4H), 7.307.17 (m, 4H), 4.92 (s, 2H), 4.37 (t, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 2.96 (m, 4H), 2.81-2.62 (m,
8H), 2.33-2.11 (m, 2H), 1.95 (q, J=7.0Hz, 1H), 1.63-1.55 (m, 8H), 1.50-1.40 (m, 2H), 1.38-1.33 (m,
4H), 1.29 (d, J=7.0Hz, 3H), 0.83 (d, J=7.0Hz, 6H). LCMS [M+H] = 844.3.
-220[0447] The following ATAC6 compound and ATAC7 compound in TABLE 3 can be prepared using a method similar to that described above for ATAC5.
TABLE 3
Compound Structure Name M+l
ATAC6 ° z 2,5-dioxopyrrolidin-l-yl 7- (((S)-1-(((S)-1-((4-(((((4- amino-1 -(2-hydroxy-2 - methylpropyl)- lH-imidazo [4,5 - c]quinolin-2- yl)methyl)(ethyl)carbamoyl)ox y)methyl)phenyl)amino) -1 - oxopropan-2-yl)amino)-3 - methyl-1 -oxobutan-2- yl)amino)-7-oxoheptanoate 872
ATAC7 h2n^o HN 2,5-dioxopyrrolidin-l-yl 7- (((S)-1-(((S)-1-((4-(((((4- amino-1 -(2-hydroxy-2 - methylpropyl)- lH-imidazo [4,5 - c]quinolin-2- yl)methyl)(ethyl)carbamoyl)ox y)methyl)phenyl)amino)-1 -oxo- 5 -ureidopentan-2-yl)amino)-3 - methyl-1 -oxobutan-2- yl)amino)-7-oxoheptanoate 958
EXAMPLE 7
Synthesis of ATAC8, ATAC9, and ATAC10 [0448] This example shows synthesis of Perfluorophenyl 6-(((S)-l-(((S)-l-((4-((((5-(2-amino-3pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-3methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC8), perfluorophenyl 7-(((S)-l-(((S)-l-((4(((((4-amino-1 -(2-hydroxy-2-methylpropyl)-1 H-imidazo[4,5 -c] quinolin-2yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC9), and perfluorophenyl 7-(((S)-l-(((S)-l-((4-(((((4 amino-l-(2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2-221yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC10).
Figure GB2552041A_D0279
Step A: Preparation of ATAC8
Figure GB2552041A_D0280
[0449] To a stirred solution of 6-(((S)-l-(((S)-l-((4-((((5-(2-amino-3-pentylquinolin-5yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 -oxobutan-2yl)amino)-6-oxohexanoic acid hydrochloride (168 mg, 0.215 mmol) and pentafluorophenol (158 mg,
0.86 mmol) in DMF (3 ml) was added Ν,Ν'-diisopropylcarbodiimide (0.166 ml, 1.07 mmol) dropwise and the reaction mixture was stirred at room temperature for 6h. The reaction mixture was concentrated and the residue was dissolved in DMSO and purified by reverse phase chromatography [water/acetonitrile (0.1% TFA)] from 10% followed by a gradient from 20 to 80%. Pure fractions were combined to give perfluorophenyl 6-(((S)-l-(((S)-l-((4-((((5-(2-amino-3-pentylquinolin-5yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1 -oxopropan-2-yl)amino)-3 -methyl-1 -oxobutan-2yl)amino)-6-oxohexanoate 2,2,2-trifluoroacetate (122 mg) as a white solid. M NMR (DMSO-d6) δ
13.5 (s, 1H), 9.92 (s, 1H), 8.35 (bs, 3H), 8.17 (d, J=7.0Hz, 1H), 7.87 (d, J=7.0Hz, 1H), 7.64-7.52 (m,
4H), 7.32-7.18 (m, 4H), 4.91 (s, 2H), 4.37 (t, J=7.0Hz, 1H), 4.19 (t, J=7.0Hz, 1H), 3.60-3.50 (m, 4H)
-2222.97 (m, 4H), 2.79 (t, J=7.0Hz, 2H), 2.74 (t, J=7.0Hz, 2H), 2.31-2.22 (m, 2H), 1.96 (q, J=7.0Hz, 1H), 1.71-1.51 (m, 8H), 1.45-1.38 (m, 2H), 1.40-1.27 (m, 9H), 0.90-0.80 (m, 9H). LCMS [M+H] = 913.4. [0450] The following compound in TABLE 4 can be prepared using a method similar to that described in above for ATAC8.
TABLE 4
Compound Structure Name M+l
ATAC9 F H i? fHa H H4 FIX ° ° H ° ΥτΥγ) F 0 Τ Hof perfluorophenyl 7-(((S)-l-(((S)- 1 -((4-(((((4-amino-1-(2- hydroxy-2-methylpropyl)- 1H- imidazo [4,5 -c]quinolin-2- yl)methyl)(ethyl)carbamoyl)oxy )methyl)phenyl)amino)-1 - oxopropan-2-yl)amino)-3 - methyl-1 -oxobutan-2-yl)amino)- 7-oxoheptanoate 941
ATAC10 HjNyO HN, .................... F 0 ho^ perfluorophenyl 7-(((S)-l-(((S)- 1 -((4-(((((4-amino-1-(2- hydroxy-2-methylpropyl)- 1H- imidazo [4,5 -c]quinolin-2- yl)methyl)(ethyl)carbamoyl)oxy )methyl)phenyl)amino)-1 -oxo-5 - ureidopentan-2-yl)amino)-3- methyl-1 -oxobutan-2-yl)amino)- 7-oxoheptanoate 1027
EXAMPLE 8 Synthesis of ATAC11 [0451] This example shows the synthesis ofN-((4-amino-l-(2-hydroxy-2-methylpropyl)-lHimidazo[4,5-c]quinolin-2-yl)methyl)-l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-Nethyl-3,6,9,12-tetraoxapentadecan-15-amide (ATAC11).
-223ο ο ο
Figure GB2552041A_D0281
ATAC11
Step A: Preparation of ATAC 11
Figure GB2552041A_D0282
ATAC11 [0452] A solution of MAL-PEG4-acid (265.7 mg, 0.638 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 144.8 mg, 0.702 mmol) in dry di chloromethane / acetonitrile (1:1, 5 mL) was stirred at room temperature for lh, followed by addition of compound 1 (100 mg, 0.319 mmol) in one portion. After 72h of stirring, volatile organics were removed under vacuum. The residue obtained was purified by flash column chromatography on silica gel, eluting with step gradients of methanol in dichloromethane at a ratio of v/v 1:20, 1:15, and 1:9, to afford the target product N-((4-amino-1-(2hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2-yl)methyl)-l-(3-(2,5-dioxo-2,5-dihydro-lHpyrrol-l-yl)propanamido)-N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (80 mg, 35% yield) as white colored foamy solid oil. *HNMR (300 MHz, CDC13) δ 8.40 - 7.82 (br m, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.44 (t, J=7.5 Hz, 1H), 7.30 (t, J=7.4 Hz, 1H), 6.76 - 6.28 (br m, 2H), 4.82 - 4.32 (br m, 2H), 4.08 - 3.64 (br m, 6H), 3.54 (br s, 14H), 3.31 (br s, 3H), 2.63 (br s, 2H), 2.38 (t, J=6.9 Hz, 2H), 1.27 (br s, 4H), 1.20 - 0.68 (br m, 5H). MS (ESI+) m/z 712 (M+l), 734 (M+Na).
EXAMPLE 9
Synthesis of ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19,
ATAC20, and ATAC21
-224[0453] This example shows the synthesis of N-(5-(2-amino-3-pentylquinolin-5-yl)pentyl)-l-(3-(2,5dioxo-2,5 -dihydro-1 H-pyrrol-1 -yl)propanamido)-3,6,9,12-tetraoxapentadecan-15 -amide (ATAC 12),
1- (3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-(3-pentylquinolin-2-yl)-3,6,9,12tetraoxapentadecan-15-amide (ATAC13), l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)-N-(l-isobutyl-lH-imidazo[4,5-c]quinolin-4-yl)-3,6,9,12-tetraoxapentadecan-15amide (ATAC14), 1-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-methyl-N-(2-(3-(7methylbenzo[l,2-d:3,4-d']bis(thiazole)-2-yl)ureido)ethyl)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC15), (S)-l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-N-(l-((7methylbenzo[l,2-d:3,4-d']bis(thiazole)-2-yl)amino)-l-oxo-3-phenylpropan-2-yl)-3,6,9,12tetraoxapentadecan-15-amide (ATAC 16), N-(benzo[d]thiazol-2-yl)-1 -(3-(2,5-dioxo-2,5-dihy dro- 1H pyrrol-l-yl)propanamido)-N-((8-hydroxyquinolin-7-yl)(4-(trifluoromethoxy)phenyl)methyl)3,6,9,12-tetraoxapentadecan-15-amide (ATAC17), N-((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9bis(2-amino-6-oxo-lH-purin-9(6H)-yl)-5,10,12-trihydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'j][l,3,7,9,2,8]tetra-oxadiphosphacyclododecin-3-yl)-l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)propanamido)-3,6,9,12-tetraoxapentadecan-l5-amide (ATAC18), N((2R,3R,3 aS,7aR,9R, 1 OR, 1 OaS, 14aR)-2,9-bis(2-amino-6-oxo- lH-purin-9(6H)-yl)-10-hydroxy-5,12dimercapto-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-j][l,3,7,9,2,8]tetraoxadiphosphacyclododecin3-yl)-l-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15amide (ATAC 19), N-(9-((2R,3R,3aS,7aR,9R, 1 OR, 1 OaS, 14aR)-9-(2-amino-6-oxo-lH-purin-9(6H)yl)-3,5,10,12-tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-j][l,3,7,9,2,8]tetraoxadiphosphacy clododecin-2-yl)-9H-purin-6-yl)-1 -(3 -(2,5 -dioxo-2,5-dihy dro-1 H-pyrrol-1 yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC20), and N-(9((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-9-(2-amino-6-oxo-lH-purin-9(6H)-yl)-3,5,10,12tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3', 2'-j][l, 3,7,9,2,8]tetraoxadiphosphacyclododecin2- yl)-9H-purin-6-yl)-1-(3 -(2,5-dioxo-2,5 -dihydro-1 H-pyrrol-1 -yl)propanamido)-3,6,9,12tetraoxapentadecan-15-amide (ATAC21).
Figure GB2552041A_D0283
-225Step A: Preparation of ATAC 12
Figure GB2552041A_D0284
DCM
Figure GB2552041A_D0285
nh2
ATAC12 [0454] To a stirred solution containing 100 mg (0.33 mmol) of 5-(5-aminopentyl)-3-pentylquinolin2-amine in 13 mL of CH2CI2 under N2 was added a solution of MAL-PEG4-NHS [CAS No 75652599-2] (171 mg, 0.33 mmol) in 3 mL of CH2CI2 by syringe pump over 90 mins. The reaction mixture was stirred at room temperature for 16h then evaporated to afford a residue which was purified by silica gel chromatography (CombiFlash Gold (12g): CH2CI2/CH3OH/NH4OH) to afford a light yellow syrup which was dissolved in 5 mL of CH3CN and lyophilized to provide 164 mg of the desired compound. *HNMR (CD3OD) δ 7.95 (s, 1H), 7.38 (s, 1H), 7.37 (s, 1H), 7.07 (t, J=8.5Hz,
1H), 6.78 (s, 2H), 3.75 (t, J=6.0Hz, 2H), 3.65 (t, J=6.0Hz, 2H), 3.59-3.52 (m, 12H), 3.46 (t, J=5.5Hz, 2H), 3.28 (t, J=7.5Hz, 2H), 3.18 (t, J=7.5Hz, 2H), 2.98 (t, J=8.5Hz, 2H), 2.67 (t, J=7.5Hz, 2H), 2.44 (t, J=7.0 Hz, 2H), 2.40 (t, J=7.0 Hz, 2H), 1.76-1.68 (m, 4H), 1.58-1.52 (m, 2H), 1.46-1.40 (m, 6H), 0.94 (t, J=7.0Hz, 3H). (MS (ESI+) m/z 698 (M+l).
[0455] The following compounds in TABLE 5 can be prepared using a method similar to that as described above for ATAC 12.
TABLE 5
Compound Structure Name M+l
ATAC 13 0 0 O N |[ H H H J ° 1 1 -(3 -(2,5 -dioxo-2,5 -dihydro- 1H- pyrrol-1 -yl)propanamido) -N-(3 - pentylquinolin-2-yl)-3,6,9,12- tetraoxapentadecan-15 -amide 613
-226-
ATAC14 ο Ο Ο Ν |[ VI Η Η Ά A χ0 ζ 1 -(3 -(2,5 -dioxo-2,5 -dihydro- 1H- pyrrol-1 -yl)propanamido) -N-( 1 - isobutyl- lH-imidazo [4,5 - c]quinolin-4-yl)-3,6,9,12- tetraoxapentadecan-15 -amide 639
ATAC15 ΛνΧν^°^ο^°^ oXAus0v™= V4o Η X 0 W^AA^-S 1 -(3 -(2,5 -dioxo-2,5 -dihydro- 1H- pyrrol-1 -yl)propanamido) -N - methyl-N-(2-(3-(7- methylbenzo [ 1,2-d: 3,4- d']bis(thiazole)-2-yl)ureido)ethyl)- 3,6,9,12-tetraoxapentadecan-15- amide 720
ATAC16 R η H1 NyS ch3 (S)-1 -(3 -(2,5 -dioxo-2,5 -dihydro- lH-pyrrol-1 -yl)propanamido)-N- (1-((7-methylbenzo [ 1,2-d: 3,4- d']bis(thiazole)-2-yl)amino)-l- oxo-3 -phenylpropan-2 -yl) - 3,6,9,12-tetraoxapentadecan-15- amide 635
ATAC17 P ° i? Xs PA MMvtW V—X Ο A. OH ocf3 N-(benzo [d]thiazol-2 -yl)-1-(3- (2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)propanamido)-N-((8- hydroxyquinolin-7-yl)(4- (trifluoromethoxy)phenyl)methyl)- 3,6,9,12-tetraoxapentadecan-15- amide 734
ATAC18 ο <X HO0 00 Ηΐγ X Η,ν AX ΑγΧ? 0 ° 0 N- ((2R,3R,3aS,7aR,9R, 10R, lOaS, 14 aR)-2,9-bis(2-amino-6-oxo-lH- purin-9(6H)-yl)-5,10,12- trihydroxy-5,12- dioxidodecahydrodifuro [3,2- d:3',2'-j][l,3,7,9,2,8]tetra- oxadiphosphacyclododecin-3 -yl)- 1 -(3 -(2,5 -dioxo-2,5 -dihydro- 1H- 1088
-227-
pyrrol-1 -yl)propanamido)- 3,6,9,12-tetraoxapentadecan-15- amide
ATAC19 0 ^'X'nh HSP // Η/ / ο /rt ° Υ HN/°——ο/γ-Λ ΗΝγ/ ° 0 N- ((2R,3R,3aS,7aR,9R, 10R, lOaS, 14 aR)-2,9-bis(2-amino-6-oxo-lH- purin-9(6H)-yl)-10-hydroxy-5,12- dimercapto-5,12- dioxidodecahydrodifuro [3,2- d:3',2'- j] [ 1,3,7,9,2,8]tetraoxadiphosphacy clododecin-3 -yl)-1 -(3 -(2,5 -dioxo- 2,5 -dihydro-1 H-pyrrol-1 - yl)propanamido)-3,6,9,12- tetraoxapentadecan-15 -amide 1120
ATAC20 0 o ο X ° o <nx H y ho. y γ/κΑ χ X ΓΌ/ OHO «2Νγ,Ν ,1 °'-/ hnAG ό'0Η 0 N-(9- ((2R,3R,3aS,7aR9R 10R lOaS, 14 aR)-9-(2-amino-6-oxo-lH-purin- 9(6H)-yl)-3,5,10,12-tetrahydroxy- 5,12-dioxidodecahydrodifuro [3,2- d:3',2'-j][l,3,7,9,2,8]tetra- oxadiphosphacyclododecin-2-yl)- 9H-purin-6-yl)-1 -(3 -(2,5 -dioxo- 2,5 -dihydro-1 H-pyrrol-1 - yl)propanamido)-3,6,9,12- tetraoxapentadecan-15 -amide 1073
ATAC21 ο 0 υ HN X/^O ''/'''O-^''''-./'0 '-/''['.J -Y'/'N X N X H jX HO. y N'T/ /CO 1 χ X SoX 0 OH XNX °x4h HN^JJ-X ° 0 N-(9- ((2R,3R,3aS,7aR9R 10R lOaS, 14 aR)-9-(2-amino-6-oxo-lH-purin- 9(6H)-yl)-3,5,10,12-tetrahydroxy- 5,12-dioxidodecahydrodifuro[3,2- d:3',2'- j] [ 1,3,7,9,2,8]tetraoxadiphosphacy clododecin-2-yl)-9H-purin-6-yl)- 1 -(3 -(2,5 -dioxo-2,5 -dihydro- 1H- 1073
-228-
pyrrol-1 -yl)propanamido)- 3,6,9,12-tetraoxapentadecan-15- amide
EXAMPLE 10
Synthesis of ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, and ATAC31 [0456] This example shows the synthesis of 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)hexanamido)-3-methylbutanamido)propanamido)benzyl ((4-amino-1 -(2-hydroxy-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate (ATAC22), 4-((S)-2-((S)-2-(6(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)-3-methyl-butanamido)propanamido)benzyl (5(2-amino-3-pentylquinolin-5-yl)pentyl)-carbamate (ATAC23), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5dihydro-lH-pyrrol-l-yl)hexanamido)-3-methylbutan-amido)-5-ureidopentanamido)benzyl-(5-(2amino-3-pentylquinolin-5-yl)pentyl)-carbamate (ATAC24), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5dihydro-lH-pyrrol-l-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl((4-aminol-(2-hydroxy-2-methylpropyl)- lH-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate TFA salt (ATAC25), 2-(3-{2-[N-Methyl({p-[(S)-2-{(S)-2-[6-(2,5-dioxo-lH-pyrrol-l-yl)hexanoylamino]-3methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino]ethyl}ureido)-7methyl-1,6-dithia-3,8-diaza-as-indacene (ATAC26), 2-{[(8-Hydroxy-7-quinolyl)(ptrifluoromethoxyphenyl)methyl]({p-[(S)-2-{(S)-2-[6-(2,5-dioxo-lH-pynOl-l-yl)hexanoylamino]-3methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino}-l,3-benzothi azole (ATAC27), (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-lyl)hexanoylamino]-3-methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonylamino)8.17- bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-3,12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa3λ5.12λ5-diphosphatricyclo[ 13.3.0.06,10]octadecane-3,12-dione (ATAC28), (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-lyl)hexanoylamino]-3-methylbutyrylamino}propionylamino]phenyl}methoxycarbonylamino)-8,17bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-3,l 2-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa3λ5.12λ5-diphosphatricyclo[ 13.3.0.06,10]octadecane-3,12-dione (ATAC29), (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-lyl)hexanoylamino]-3-methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonylamino)8.17- bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-9-hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa
-2293Z5.12X5-diphosphatricyclo[13.3.0.06,10]octadecane-3,12-dione (ATAC30), and {p-[(S)-2-{(S)-2[6-(2,5-Di oxo-lH-pyrrol-1-yl)hexanoylamino]-3-methylbutyrylamino}-5ureidovalerylamino]phenyl}methyl 9-{(lS,6R,8R,9S,10S,15R,17R,18S)-8-(2-amino-6-oxo-l,9dihydropurin-9-yl)-3,12-dihydroxy-9,18-dihydroxy-3,12-dioxo-2.4.7.11.13.16-hexaoxa-3Z5.12Z5diphosphatricyclo[13.2.1.06,10]octadec-17-yl}-9a-adenineecarboxylate (ATAC31).
Figure GB2552041A_D0286
ATAC22
Step A: Preparation of ATAC22
Figure GB2552041A_D0287
ATAC22 [0457] A solution of compound 1 (150 mg, 0.479 mmol) and Ν,Ν’-diisopropylethylamine (145.4 mg, 1.437 mmol) in dry DMF was stirred at room temperature for 5 min., followed by addition of maleimidocaproyl-valine-alanine-p-aminobenzyl alcohol p-nitrophenyl-carbonate (MC-Val-AlaPAB-PNP, 343.6 mg, 0.527 mmol). After stirring for 24 h, volatile organics were removed under vacuum. The residue obtained was triturated with dry acetonitrile. The precipitated solid was collected by filtration, washed with acetonitrile and dried under vacuum to obtain unreacted MCVal-Ala-PAB-PNP (130 mg) as beige solid. The filtrate and washings were combined and concentrated under vacuum. The residue obtained was purified by flash column chromatography on silica gel, eluting with step gradients of MeOH in di chloromethane at a ratio of v/v 1:20, 1:15, and
-2301:10, to afford the target product mc-Val-Ala-PAB-GDQ (70 mg, 18% yield) as beige colored foamy solid. 'H NMR (DMSO-d6) δ 10.1 -9.75 (brm, 1H), 8.58 - 8.24 (br m, 1H), 8.15 (d, J=6.6 Hz, 1H), 8.01 (br s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.4 Hz, 1H), 7.65 - 7.48 (m, 2H), 7.46 - 7.34 (m, 2H), 7.29 (br s, 1H), 7.18 (br s, 1H), 6.99 (s, 2H), 5.03 (br s, 2H), 4.96 (br s, 1H), 4.72 (br s, 1H), 4.48-4.26 (m, 1H), 4.26 - 4.04 (m, 1H), 2.22 - 2.02 (m, 2H), 2.02 - 1.80 (m, 1H), 1.58 - 1.37 (m, 4H), 1.36 - 0.92 (br m, 15H), 0.92 - 0.53 ( br m, 7H). MS (ESI+) m/z 826 (M+l).
[0458] The following ATAC30, ATAC31, ATAC32, ATAC33, ATAC34, ATAC35, ATAC36, ATAC37, ATAC38, ATAC39, ATAC40, ATAC41, and ATAC42 can be prepared using a method similar to that described above for ATAC29.
ATAC23: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl (5-(2-amino-3-pentylquinolin-5-yl)pentyl)-carbamate
Figure GB2552041A_D0288
ATAC23 [0459] *HNMR (CD3OD) δ 8.35 (s, 1H), 7.63 (t, J=8.5Hz, 1H), 7.55 (d, J=8.0Hz, 1H), 7.47 (d, J=8.0Hz, 1H), 7.33 (d, J=8.0Hz, 1H), 7.27(d, J=8.0Hz, 1H), 6.78 (s, 2H), 5.00 (s, 2H), 4.46 (q, J=7.0Hz, 2H), 4.13 (d, J=7.0Hz, 1H), 3.47-3.4 (m, 3H), 3.17 (t, J=7.0Hz, 2H), 3.05 (t, J=7.0Hz, 2H), 2.75 (t, J=7.5Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 2.07 (q, J=7.0Hz, 1H), 1.72-1.51 (m, 10H), 1.46-1.35 (m, 8H), 1.32-1.26 (m, 3H), 1.00-0.92 (m, 9H). LCMS [M+H] = 812.4.
ATAC24: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl-(5-(2-amino-3-pentylquinolin-5-yl)pentyl)-carbamate
-231-
Figure GB2552041A_D0289
H2nXq
ATAC24 'H NMR (DMSO-d6) δ 13.5 (bs, 1H), 10.0 (s, 1H), 8.40 (m, 3H), 8.07 (d, J=7.5Hz, 1H), 7.80 (d, J=8.5 Hz, 1H), 7.6-7.5 (m, 4H), 7.35-7.25 (m, 2H), 6.01 (m, 1H), 5.42 (s, 1H), 4.89 (s, 2H), 4.41 (q, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 3.10 - 2.90 (m, 6H), 2.75 (t, J=7.5Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 2.07 (q, J=7.0Hz, 1H), 1.72-1.51 (m, 10H), 1.46-1.35 (m, 8H), 1.32-1.26 (m, 3H), 1.00-0.92 (m, 9H). LCMS [M+H] = 898.
ATAC25: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)-3methylbutanamido)-5-ureidopentanamido)benzyl((4-amino-1 -(2-hydroxy-2-methylpropyl)- 1Himidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate TFA salt
Figure GB2552041A_D0290
ATAC25 *HNMR (DMSO-d6) δ 13.4 (bs, 1H), 9.99 - 9.89 (br m, 1H), 9.09 - 8.40 (m, 3H), 8.07 (d, J=7.5Hz, 1H), 7.80 (d, J=8.5 Hz, 1H), 7.68 (t, J=8.0 Hz, 1H), 7.59 (bs, 1H), 7.51 (t, J=8.5Hz, 1H), 7.46 - 7.14 (m, 2H), 7.00 (s, 1H), 5.99 (br s, 1H), 5.05 (br s, 1H), 4.95 (br s, 1H), 4.37 (q, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 3.37 (t, J=7.0Hz, 2H), 3.03 - 2.93 (m, 2H), 2.22 - 2.07 (m, 2H), 1.99 - 1.92 (m, 1H), 1.75 - 1.05 (br m, 20H), 0.85 (d, J=8.5Hz, 3H), 0.81 (d, J=8.5Hz, 3H). MS (ESI+) m/z 912.5 (M+l).
-232-
TABLE 6
Compoun d Structure Name M+l
ATAC26 o H 0 Y H ^NH (Am, 2-(3-{2-[N-Methyl({p-[(S)-2-{(S)-2-[6-(2,5- dioxo-1 H-pyrrol-1 -yl)hexanoylamino] -3 - methylbutyrylamino } -5 - ureidovalerylamino]phenyl}methoxy carbonyl )amino] ethyl} ureido)-7-methyl-1,6-dithia-3,8 - diaza-as-indacene 921
ATAC27 ocf3 S H 8 cH AJ β o^nh2 2-{[(8-Hydroxy-7-quinolyl)(p- trifluoromethoxyphenyl)methyl]({p-[(S)-2- {(S)-2-[6-(2,5-dioxo-lH-pyrrol-l- yl)hexanoylamino] -3 -methylbutyrylamino } -5 - ureidovalerylamino]phenyl}methoxy carbonyl )amino} -1,3 -benzothiazole 1067
ATAC28 0 h2V Ho.O N NH2 hn Hi° Q/P °V°-J \ r°V /%n rTNZNVY^^N6 η’νΥνγν °Joh ° HAo A> hnZ4 0 0 (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)- 2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-l- yl)hexanoylamino] -3 -methylbutyrylamino } -5 - ureidovalerylamino]phenyl}methoxy carbonyl amino)-8,17 -bis(2-amino-6-oxo-1,9- dihydropurin-9-yl)-3,12-dihydroxy-9- hydroxy-2.4.7.11.13.16-hexaoxa-3/.5.12λ5 - diphosphatricyclo [13.3.0.06,1 Ooctadecane- 3,12-dione 1289
ATAC29 <'Λη HO 0 N'^'NH2 .'p-cl .....va.........z ΗΝγ1/ ° 0 (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)- 2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-l- yl)hexanoylamino] -3 - methylbutyrylamino } propionylamino] phenyl} methoxycarbonylamino)-8,17-bis(2-amino-6- oxo-l,9-dihydropurin-9-yl)-3,12-dihydroxy-9- hydroxy-2.4.7.11.13.16-hexaoxa-3 λ5.12λ5 - diphosphatricyclo [13.3.0.06,1 Ooctadecane- 3,12-dione 1203
-233-
ATAC30 0 HS.P ? N NH2Hn/ h\ o H2NYnJ °TsH V-W 0 HAo XX ΗΝγΜ ° 0 (lR,6R,8R,9S,10S,15R,17R,18S)-18-({p-[(S)- 2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-l- yl)hexanoylamino] -3 -methylbutyrylamino } -5 - ureidovalerylamino]phenyl}methoxy carbonyl amino)-8,17 -bis(2-amino-6-oxo-1,9- dihydropurin-9-yl)-9-hydroxy-3,12- dimercapto-2.4.7.11.13.16-hexaoxa-3 75.1275 - diphosphatricyclo [13.3.0.06,10]octadecane- 3,12-dione 1321
ATAC31 HN^O'^YZ 0 η Z' J Z/ <NZ XYiB 1 HO P Y / P-O,, HN ηϊ° v°J >—< >—r h2n^o r°A JTo h2nyn ' °X0H ΗΝγ+Ν 0 {p-[(S)-2-{(S)-2-[6-(2,5-Dioxo-lH-pyrrol-l- yl)hexanoylamino] -3 -methylbutyrylamino } -5 - ureidovalerylamino]phenyl}methyl 9- {(lS,6R,8R,9S,10S,15R,17R,18S)-8-(2- amino-6-oxo-l ,9-dihydropurin-9-yl)-3,12- dihydroxy-9,18-dihydroxy-3,12-dioxo- 2.4.7.11.13.16-hexaoxa-3 75.1275 - diphosphatricyclo [13.2.1.06,10] octadec-17- yl} -9a-adenineecarboxylate 1274
EXAMPLE 11 Synthesis of ATAC32 [0460] This example shows the synthesis of l-{6-[({7-Amino-3-(2-hydroxy-2-methylpropyl)-3.5.8triazatricyclo[7.4.0.02,6]trideca-l(9),2(6),4,7,10,12-hexaen-4-yl}methyl)-N-ethylamino]-6oxohexyl} -1 H-pyrrole-2,5 -dione (ATAC32).
Figure GB2552041A_D0291
Step A: Preparation of ATAC32
-234ο
Figure GB2552041A_D0292
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Figure GB2552041A_D0293
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ATAC32 [0461] To an ice-cold solution of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoic acid (0.034 g, 0.16 mmol) in DCM (0.800 ml) was added l-chloro-N,N,2-trimethylprop-l-en-l-amine (0.021 mL,
0.160 mmol) dropwise. This was stirred at 0°C for lh then added to an ice-cold mixture of compound 1 (50 mg, 0.160 mmol) and triethylamine (66.7 pL, 0.479 mmol) in DCM (800 pL). Overall molarity 0.1 M. The mixture was stirred to room temperature overnight and then chromatographed (DCM to 20% MeOH/DCM) without work-up. Fractions containing product were pooled and evaporated then dissolved in 1 mL of acetonitrile and treated with 0.1 mL of trifluoroacetic acid. The resulting material was evaporated to an oil then redissolved in CH3CN and lyophilized the sample to give ATAC32 (65 mg) as a white solid. Ή NMR (400 MHz, (DMSO-d6) δ 13.3 (s, 1H), 8.54 - 8.50 (m, 3H), 7.81 (d, J=8.5 Hz, 1H), 7.76 (d, J=7.5 Hz, 1H), 7.51 (d, J=7.5 Hz, 1H), 6.99 (s, 1H), 6.95 (s, 1H), 3.51 (q, J=7.0Hz, 2H), 3.43-3.31 (m, 3H), 2.36-2.30 (m, 2H),
1.54-1.41 (m, 4H), 1.25-1.00 (m, 10H). 19F NMR (DMSO-d6) δ-74.0. LCMS [M + H]+= 507.1.
EXAMPLE 12
Synthesis of ATAC33 [0462] This example shows the synthesis of l-{[4-({6-[({7-Amino-3-(2-hydroxy-2-methylpropyl)3.5.8-triazatricyclo[7.4.0.02,6]trideca-l(9),2(6),4,7,10,12-hexaen-4-yl}methyl)-N-ethylamino]-6oxohexylamino}carbonyl)cyclohexyl]methyl}-lH-pyrrole-2,5-dione (ATAC33).
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Figure GB2552041A_D0294
Step A: Preparation of ATAC33
ATAC33
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Figure GB2552041A_D0295
ATAC33 [0463] To a stirred solution of l-(4-amino-2-((ethylamino)methyl)-lH-imidazo[4,5-c]quinolin-l-yl) 2-methylpropan-2-ol (100 mg, 0.319 mmol) in DCM (10 mL) under nitrogen was added via a syringe pump a solution of 2,5-dioxopyrrolidin-l-yl 6-(4-((2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)methyl)cyclohexane-l-carboxamido)hexanoate (143 mg, 0.319 mmol) in DCM (5 mL) over a period of 3.5 h. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by reverse phase column chromatography. Pure fractions identified by HPLC analysis were pooled and concentrated. The residue was lyophilized from CH3CN to provide a white solid (52.8, mg) as the TFA salt of ATAC33 as a mixture of cis and trans isomers. Ή NMR (400 MHz, (CD3OD) δ 8.54 and 8.48 (d, J=8.3 Hz, 1H), 7.81-7.71 (m, 2H), 7.62-7.55 (m, 1H), 6.80 (s, 2H), 3.66 (q, J=7.0 Hz, 2H), 3.13 and 3.08 (t, J=7.0 Hz, 2H), 2.45 and 2.38 (t, J=7.5 Hz, 2H), 2.1-2.0 (m, 1H), 1.8-1.47 (m, 10H), 1.46-1.15 (m, 16H), 1.54-1.41 (m, 4H), 1.25-1.00 (m, 10H). LCMS [M + H]+= 646.3.
EXAMPLE 13 Synthesis of ATAC34 [0464] This example shows the synthesis of l-[(4-{[({7-Amino-3-(2-hydroxy-2-methylpropyl)3.5.8-triazatricyclo[7.4.0.02,6]trideca-l(9),2(6),4,7,10,12-hexaen-4-yl}methyl)-N-ethylamino]carbonyl}cyclohexyl)methyl]-lH-pyrrole-2,5-dione (ATAC34).
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Figure GB2552041A_D0296
Step A: Preparation of ATAC34
Figure GB2552041A_D0297
Figure GB2552041A_D0298
[0465] To an ice-cold solution of (lr,4r)-4-((2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)methyl)cyclohexane-l-carboxylic acid (82 mg, 0.346 mmol) in DCM (1728 μΐ) was added 1chloro-N,N,2-trimethylprop-l-en-l-amine (50.3 pL, 0.380 mmol) dropwise. This was stirred at 0°C for lh then added to an ice-cold mixture of l-(4-amino-2-((ethylamino)methyl)-lH-imidazo[4,5c]quinolin-l-yl)-2-methylpropan-2-ol (100 mg, 0.319 mmol) and triethylamine (133 pi, 0.957 mmol) in 1.6 mL of DCM. The mixture became a yellow solution as it stirred overnight to room temp. The reaction was concentrated to dryness, redissolved in MeOH/CLLCh, silica gel was added, then the solvents evaporated. Chromatography (12 g Gold silica, DCM to 20% MeOH/DCM, dry load) gave a solid which was dissolved in CH3CN, frozen and lyophilized to afford 170 mg of N-((4-((l(dimethylamino)-2-methylprop-1 -en-1 -yl)amino)-1 -(2-((1 -(dimethylamino)-2-methyl-prop-1 -en-1 yl)oxy)-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-2-yl)methyl)-4-((2,5-dioxo-2,5-dihydro-lHpyrrol-l-yl)methyl)-N-ethylcyclohexane-l-carboxamide which was subsequently dissolved in 50% aqueous MeCN containing 0.1%TFA and heated in a microwave reactor at 150°C for 60 min. The reaction mixture was cooled and the solvents were evaporated and chromatographed to give ATAC34 (72 mg) as a white solid. *HNMR (400 MHz, (DMSO-d6) δ 13.3 (s, 1H), 8.70 - 8.50 (m, 3H), 7.83-7.79 (m, 1H), 7.71-7.65 (m, 1H), 7.55-7.48 (m, 1H), 7.00 (s, 1H), 6.98 (s, 1H), 5.13 (bs, 1H), 4.83 (bs, 1H), 3.65 (q, J=7.0 Hz, 2H), 3.38 (m, 1H),3.25 and 3.18 (d, J=6.5 Hz, 2H), 1.69-1.52 (m, 5H), 1.45-0.88 (m, 13H). 19F NMR (DMSO-d6) δ -73.7. LCMS [M + H]+= 533.1.
-237EXAMPLE 14
Fc Receptor Binding to anti-CD40 Antibody Immune-Stimulatory Compound Conjugates [0466] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody. An anti-CD40 antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains from a SBT040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-CD40 antibody is comprised of two SBT-040-G1DE heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is comprised of two SBT040-G1 AAA heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-AAA antibody.
[0467] Each antibody is purified and then each is conjugated to ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6,
ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15,
ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or ATAC43 as described in EXAMPLE 2. Each of these conjugates is characterized for the ability of their Fc domains to bind to and for their affinity for soluble glycosylated FcyR ectodomains from human FcyRs. This is shown by performing surface plasmon resonance experiments. In these experiments, biotinylated soluble glycosylated FcyR ectodomains from all human FcyRs are immobilized on a streptavidin-coated surface. The ability of each conjugate to bind to soluble glycosylated FcyR ectodomains from all human FcyRs is then measured by surface plasmon resonance using a Biacore instrument. The data from these experiments shows that the Fc domain of any one of the SBT-040-WT-ATAC1 - SBT-040-WT-ATAC34 or SBT-040-ATAC43 conjugates, the Fc domain of any one of the SBT-040-VLPLL-ATAC1 - SBT-040-VLPLLATAC34 or SBT-040-VLPLL-ATAC43 conjugates, the Fc domain of any one of the SBT-040-DEATAC1 - SBT-040-DE-ATAC34 or SBT-040-DE-ATAC 43 conjugates, the Fc domain of any one of the SBT-040-AAA-ATAC1 - the SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugates is bound to soluble glycosylated FcyR ectodomains from human FcyRs. Therefore, the surface plasmon resonance experiments show that the ability of the Fc domain of the antibody component of the conjugate to bind to human FcyRs is not interfered with by the conjugation of the components of the conjugate. The affinity of each conjugate for each human FcyRs is also shown by the surface plasmon resonance experiments. These affinity measurements are compared with the affinity measurements for each antibody alone (as can be shown by EXAMPLE 1). The similarity in
-238affinity of each antibody alone for soluble glycosylated FcyR ectodomains from all human FcyRs with the affinity of each corresponding conjugate for soluble glycosylated FcyR ectodomains from all human FcyRs is shown by this comparison.
EXAMPLE 15
Affinity of anti-CD40 Antibodies to CD40 [0468] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and two light chain from a SBT-040 antibody, which are referred to as a SBT-040-WT antibody. An anti-CD40 antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains from a SBT040 antibody, which are referred to as a SBT-040-VLPLL antibody. An anti-CD40 antibody is comprised of two SBT-040-G1DE heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is comprised of two SBT040-G1 AAA heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-AAA antibody.
[0469] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and SBT-040AAA antibody are each produced by standard methods for producing antibodies. Each antibody is purified, and then is characterized for the ability to bind to CD40. This characterization is shown by experiments using flow cytometry. For these experiments, the human Burkitt’s Lymphoma tumor cell lines Raji and Daudi, which are previously shown to be CD40-positive, and the human Chronic Myelogenous Leukemia tumor cell line K562, which is previously shown to be CD40-negative are first evaluated by flow cytometry to assess their relative expression levels of CD40. This is assessed by incubating each cell line with a commercially available CD40 antibody conjugated to a fluorochrome, and then running samples of the incubation on a flow cytometer. The relative fluorescent intensity profiles for each cell line is shown by this data, indicating the level of CD40 expression of each cell line. The relative fluorescent intensity profiles of human Burkitt’s Lymphoma tumor cell lines Raji and Daudi show that CD40 is expressed in each of these cell lines, whereas the relative fluorescent intensity profile of the human Chronic Myelogenous Leukemia tumor cell line K562 show that CD40 is not expressed in the cell line. Then, each cell line is separately incubated with purified SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT040G1DE antibody, SBT-040-AAA antibody or no antibody as a control. Each incubation is further incubated with a secondary anti-human IgGl antibody conjugated with FITC, which is then each assessed by flow cytometry for the FITC fluorescent intensity profile of each sample. The ability of each antibody to detect CD40 expression on the cell lines is indicated by their FITC fluorescent
-239intensity profile. More specifically, the similarity between the SBT-040-WT antibody fluorescent intensity profile and each antibody with an Fc-enhanced IgGl isotype after incubation with each of the cell lines is shown by this data. Each Fc-enhanced IgGl isotype is not altered by the ability of the antibody to bind to CD40-positive cells is also shown by this data.
EXAMPLE 16
Affinity of anti-CD40 Antibodies to CD40 [0470] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and two light chain from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody. An anti-CD40 antibody is comprised of two SBT-040-G1 VLPLL heavy chains and two light chains from a SBT040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-CD40 antibody is comprised two SBT-040-G1DE heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is comprised two SBT-040G1 AAA heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT040-AAA antibody.
[0471] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and SBT-040AAA antibody are each produced by standard methods for producing antibodies. Each antibody is purified, and each antibody’s affinity for CD40 is measured. These affinities are measured by experiments using surface plasmon resonance. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. The ability of each antibody to bind to recombinant CD40 is then measured by surface plasmon resonance using a Biacore instrument. The data from these experiments shows that SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and SBT-040-AAA antibody are each bound to recombinant CD40. Therefore, each antibody’s ability to bind to CD40 is not interfered with by the enhanced Fc-enhanced IgGl isotypes is shown by the surface plasmon resonance data.
[0472] Furthermore, surface plasmon resonance is used to show that CD40L binding to CD40 is not blocked by these antibodies. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. Surface plasmon resonance using a Biacore instrument is then used to measure the binding affinity of CD40L in the presence of each antibody or without any antibody as a control. The binding affinity of CD40L with recombinant CD40 in presence of each antibody is shown to be the same as the binding affinity of the CD40L with recombinant CD40 in the absence of any antibody. Therefore, CD40 and CD40L binding is unaffected by the presence of SBT-040-WT antibody, SBT-040-G1VLPLL antibody, SBT-040-DE antibody, or SBT-040-AAA antibody.
-240EXAMPLE 17
Fc Receptor Binding to anti-CD40 Antibody Immune-Stimulatory Compound Conjugates [0473] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and two light chain from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody. An anti-CD40 antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains from a SBT040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-CD40 antibody is comprised of two SBT-040-G1DE heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is comprised of two SBT040-G1 AAA heavy chains and two light chains from a SBT-040 antibody, which is referred to as a SBT-040-AAA antibody.
[0474] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and SBT-040AAA antibody are each made following standard methods for antibody production. Each antibody is purified and then each is conjugated to ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or ATAC 43 are as described in EXAMPLE 2. The affinity of each conjugate for CD40 is then measured by experiments using surface plasmon resonance. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. The ability of each conjugate to bind to recombinant CD40 is then measured by surface plasmon resonance using a Biacore instrument. The data from these experiments shows that any one of the SBT-040-WT-ATAC1 - SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugates, any one of the SBT-040- VLPLL- AT AC 1 - SBT-040-VLPLLATAC34 or SBT-040-VLPLL-AT AC43 conjugates, any one of the SBT-040-DE-ATAC1 - SBT040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugates, or any one of the SBT-040-AAA-AT AC 1 the SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugates is bound to recombinant CD40. Therefore, the surface plasmon resonance experiments show that each component antibody’s ability to bind to CD40 is not interfered with by the enhanced Fc-enhanced IgGl isotypes nor the antibody conjugation to ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or ATAC43.
-241[0475] Furthermore, surface plasmon resonance is used to show that CD40L binding to CD40 is not blocked in the presence of each conjugate. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. Surface plasmon resonance using a Biacore instrument is then used to measure the binding affinity of CD40L in the presence of each conjugate or without any conjugate as a control. The binding affinity of CD40L with recombinant CD40 in presence of each conjugate is shown to be the same as the binding affinity of the CD40L with recombinant CD40 in the absence of any conjugate by these experiments. Therefore, CD40 and CD40L binding is unaffected by the presence of any one of the SBT-040-WT-ATAC1 - SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugates, any one of the SBT-040-VLPLL-AT AC 1 - SBT-040-VLPLLATAC34 or SBT-040-VLPLL-AT AC43 conjugates, any one of the SBT-040-DE-ATAC1 - SBT040-DE-ATAC34 or SB T-040-DE-AT AC43 conjugates, or any one of the SBT-040-AAA-AT AC 1 the SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugates.
EXAMPLE 18
Cytokine Production is Enhanced by anti-CD40 Antibody Immune-Stimulatory Compound Conjugates [0476] This example shows that cytokine production by dendritic cells is enhanced after administration of antibody-immune stimulatory compound conjugates in culture. In this experiment, dendritic cells (DCs) are derived from peripheral blood mononuclear cells (PBMCs). DCs are obtained by putting human PBMCs into a culture dish. The resulting adherent cells are washed with RPMI containing 10% fetal calf serum, and then are incubated for 7 days in complete medium containing 10 ng/mL IL-4 and 100 ng/mL GM-CSF. The non-adherent cells are isolated and are washed. These isolated cells are run by a flow cytometer to ensure CD1 lc expression, in which the DCs identity as DCs is confirmed by CD1 lc expression. The DCs are then incubated with either the antibodies as described in Example 1 or the conjugates as described in Example 3. More specifically, the DCs are incubated with any one of SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT040-DE antibody, SBT-040-AAA antibody, the SBT-040-WT-ATAC1 conjugate - SBT-040-WTATAC34 or SBT-040-WT-ATAC43 conjugate, the SBT-040-VLPLL-AT AC 1 conjugate - SBT040-VLPLL-AT AC3 4 or SBT-040-VLPLL-ATAC43conjugate, the SBT-040-DE-ATAC1 conjugate - SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugate, the SBT-040-AAA-AT AC 1 conjugate - the SB T-040-AAA-AT AC3 4 or SBT-040-AAA-ATAC43 conjugate, or a non-binding isotype control antibody. Each culture is then incubated for 24 hours and the supernatant of each culture is analyzed using a cytokine bead array assay. Cytokine expression levels of ΙΕΝγ, IL-8, IL-24212 and IL-2 are measured by the cytokine bead array assay. The supernatant from the culture containing the non-binding isotype control shows the level of cytokine expression is decreased as compared to the supernatant from cultures containing SBT-040-WT, SBT-040-VLPLL, SBT-040DE, or SBT-040-AAA. Additionally, the level of cytokine expression in the supernatant from cultures containing SBT-040-WT, SBT-040-VLPLL, SBT-040-DE, or SBT-040-AAA is decreased as compared to the supernatant from cultures containing any one of the SBT-040-WT-ATAC1 conjugate - SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugate, any one of the SBT-040VLPLL-ATAC1 conjugate - SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL ATAC43 conjugate, any one of the SBT-040-DE-ATAC1 conjugate -SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugate, or any one of the SBT-040-AAA-AT AC 1 conjugate -SBT-040-AAA-ATAC34 or SBT040-AAA-ATAC43 conjugate.
EXAMPLE 19
Cytokine Production by Dendritic Cells from Multiple Donors Was Enhanced by anti-CD40 Antibody Immune-Stimulatory Compound Conjugates [0477] Antibody-immune stimulatory compound conjugates enhanced immunostimulatory cytokines produced by human dendritic cells in a concentration dependent manner when added to and incubated with the cells. The human dendritic cells (DCs) from two donors were derived from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using a commercially available kit. The monocytes were cultured in RPMI containing 10% fetal calf serum for seven days in complete medium supplemented with 25ng/mL IL-4 and lOng/mL GMCSF. The media was replaced with fresh media plus cytokines on day three. On day six anti-CD40 antibody immune-stimulatory compound conjugates SBT-040-G1-ATAC23 and SBT-040-G1ATAC17 and control antibody were added to individual wells containing the dendritic cells. After 24 hours of further incubation, the supernatants were collected and the cytokines IL-6, TNFa, IL-12p70 and IL-10 produced by the dendritic cells were quantitated by electrochemiluminesence signal by multiplex ELISA using commercially available reagents and plate reader from Meso Scale Discovery. Results are shown for the immune stimulatory cytokines IL-12p70 and TNFa for dendritic cells derived from two donors. FIGURE 31A shows the concentration of IL-12p70 produced by DCs from donor 1 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1ATAC17as compared with SBT-050-WT. FIGURE 31B shows the concentration of IL-12p70 produced by DCs from donor 2 after incubation with SBT-040-Gl-ATAC23or SBT-040-G1ATAC17as compared with SBT-050-WT. FIGURE 31C shows the concentration of TNFa
-243produced by DCs from donor 1 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1ATAC17as compared with an anti-HER2 antibody. FIGURE 31D shows the concentration of TNFa produced by DCs from donor 1 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1ATAC17as compared with an anti-HER2 antibody.
EXAMPLE 20
Immunostimulatory Cytokine Secretion Is Enhanced By Anti-CD40 Antibody ImmuneStimulatory Compound Conjugates with Different Linkers and FcyR Binding [0478] Antibody-immune stimulatory compound conjugates enhanced human dendritic cells cytokine production in a concentration dependent manner when added to and incubated with the cells. The human dendritic cells (DCs) were derived from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using a commercially available kit. The monocytes were cultured in RPMI containing 10% fetal calf serum for seven days in complete medium supplemented with 25ng/mL IL-4 and lOng/mL GM-CSF. On day six anti-CD40 antibody immune-stimulatory compound conjugates and non-DC binding control antibody SBT-50 Gl and commercially available soluble CD40L were added to individual wells containing the dendritic cells. More specifically, the DCs are incubated with any one of SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040-AAA-ATAC29, SBT-040-VLPLL-ATAC29, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30, SBT-040-AAA-ATAC11, SBT-040-VLPLLATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAA-ATAC30. After 24 hours further incubation supernatants were collected and the cytokines IL-6, TNFa, IL-12p70 and IL-10 produced by the dendritic cells were quantitated by electrochemiluminesence signal by multiplex ELISA using commercially available reagents and plate reader from Meso Scale Discovery. FIGURE 32A shows the concentration of IL-12p70 produced by DCs after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040G2-ATAC4, SBT-040-G2-ATAC3, SBT-040-AAA-ATAC29, SBT-040-VLPLL-ATAC29, SBT040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT040-WT-ATAC30, SBT-040-AAA-ATAC11, SBT-040-VLPLL-ATAC 11, SBT-040-VLPLLATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAA-ATAC30 as compared with SBT-050-G2 antibody or CD40 ligand. FIGURE 32B shows the concentration of IL6 produced by DCs from donor 2 after incubation with SBT-040-WT-ATAC4, SBT-040-WTATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040-AAA-ATAC29, SBT-040-VLPLL-244ATAC29, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2ATAC12, SBT-040-WT-ATAC30, SBT-040-AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT040-VLPLL-AT AC 12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAAATAC30 as compared with SBT-050-G2 or CD40 ligand. Results are shown for the immune stimulatory cytokines IL-12p70 and IL-6. The treatment concentrations for each molecule, depicted on the x-axis from right to left, were 0.08 ug/mL, 0.310 ug/mL, 1.25 ug/mL and 5.00 ug/mL.
EXAMPLE 21
Anti-CD40 Antibody Immune-Stimulatory Compound Conjugates Increased Cell Surface Expression of Immune Activating Proteins [0479] Human dendritic cells showed increased expression of CD83, CD86, and MHC class II cell surface proteins in after treatment with anti-CD40 antibody immune-stimulatory compound conjugates. The increased expression of these surface proteins was dose dependent.
[0480] Human dendritic cells were derived from human PBMCs by isolation of CD14+ monocytes followed by culture in RPMI containing 10% fetal calf serum for seven days in complete medium supplemented with lOng/mL IL-4 and lOOng/mL GM-CSF. After three days of culture the media was removed and replaced with fresh media including cytokine supplement. On day six SBT-040WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30 or a control SBT-050-WT were added to separate aliquots of dendritic cells. After an additional 24 hour incubation the cells were collected and washed by centrifugations then stained for 30 minutes on ice using manufacturer’s recommended concentrations of commercially available anti-CD83, anti-CD86 and anti-MHC class II monoclonal antibodies conjugated to laser sensitive fluors. A separate aliquot for each treatment was stained with IgG matched isotype control antibody conjugate for the antiCD86 antibody, anti-CD83 antibody, and anti-MHC Class II antibody. After washing to remove unbound antibody-fluor molecules, the stained cells were subjected to FACS analysis using a Celesta flow cytometer (BD Biosciences) with gating on live cells. The output was analyzed by Flow Jo vl0.2 software (FlowJo LLC) and curve fit with Prism 7.01 software (GraphPad Software, Inc.). FIGURE 33A shows a dose dependent increase in CD86 expression on dendritic cells after treatment with SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT040-AAA-AT AC30 as compared to treatment a control SBT-050-WT or staining with an isotype control. FIGURE 33B shows a dose dependent increase in CD83 expression on dendritic cells after treatment SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040AAA-ATAC30 as compared to treatment a control SBT-050-WT or staining with an isotype control.
-245FIGURE 33C shows a dose dependent increase in MHC class II expression on dendritic cells after treatment with SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT040-AAA-ATAC30 as compared to treatment a control SBT-050-WT or staining with an isotype control. The graph shows plots of treatment protein concentration on the x axis versus mean fluorescence intensity for the cell surface protein on the y axis.
EXAMPLE 22
Treatment of Cancer By Administering a Conjugate [0481] This example describes treatment of cancer with a conjugate. A human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 8 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 8 is a conjugate comprising an antibody construct and an immune stimulatory compound. The antibody construct is an antibody, which contains two heavy chains as shown in gray and two light chains as shown in light gray. The antibody comprises two antigen binding sites (810 and 815), and a portion of the heavy chains contain Fc domains (805 and 820). The immune-stimulatory compounds (830 and 840) are conjugated to the antibody by linkers (860 and 870).
[0482] As another example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 9 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 9 is a conjugate comprising an antibody construct, two targeting binding domains, and two immune stimulatory compounds. The antibody construct is an antibody, which contains two heavy chains as shown in gray and two light chains as shown in light gray. The antibody comprises two antigen binding sites (910 and 915), and a portion of the heavy chains contain Fc domains (905 and 920). The immune-stimulatory compounds (930 and 940) are conjugated to the antibody by linkers (960 and 970). The targeting binding domains are conjugated to the antibody (980 and 985).
[0483] As an additional example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 10 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 10 is a conjugate comprising an antibody construct and two immune stimulatory compounds. The antibody construct contains the Fc region of an antibody with the heavy chains shown in gray, and two scaffolds as shown in light gray. The antibody construct comprises two antigen binding sites (1010 and 1015) in the scaffolds, and a portion of the heavy chains contain Fc domains (1005 and 1020). The immune-stimulatory compounds (1030 and 1040) are conjugated to the antibody construct by linkers (1060 and 1070).
-246[0484] As another example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 11 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 11 is a conjugate comprising an antibody construct, two targeting domains, and two immune stimulatory compounds. The antibody construct contains the Fc region of an antibody with the heavy chains shown in gray, and two scaffolds as shown in light gray. The antibody construct comprises two antigen binding sites (1110 and 1115) in the scaffolds, and a portion of the heavy chains contain Fc domains (1105 and 1120). The immune-stimulatory compounds (1130 and 1140) are conjugated to the antibody construct by linkers (1160 and 1170). The targeting binding domains are conjugated to the antibody construct (1180 and 1185).
[0485] As another example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 12 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 12 is a conjugate comprising an antibody construct and two immune stimulatory compounds. The antibody construct contains the F(ab')2 region of an antibody with heavy chains shown in gray and light chains shown in light gray, and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1210 and 1215), and a portion of two scaffolds contain Fc domains (1220 and 1245). The immune-stimulatory compounds (1230 and 1240) are conjugated to the antibody construct by linkers (1260 and 1270).
[0486] As another example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 13 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 13 is a conjugate comprising an antibody construct, two targeting binding domains, and two immune stimulatory compounds. The antibody construct contains the F(ab')2 region of an antibody with heavy chains shown in gray and light chains shown in light gray, and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1310 and 1315), and a portion of two scaffolds contain Fc domains (1320 and 1345). The immune-stimulatory compounds (1330 and 1340) are conjugated to the antibody construct by linkers (1360 and 1370). The targeting binding domains are conjugated to the antibody construct (1380 and 1385).
[0487] As another example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 14 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 14 is a conjugate comprising an antibody construct, and two immune stimulatory compounds. The antibody construct contains two scaffolds as shown in light gray and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1410 and 1415), and a portion of the two dark gray scaffolds contain Fc domains (1420 and 1445). The immune-247stimulatory compounds (1430 and 1440) are conjugated to the antibody construct by linkers (1460 and 1470).
[0488] As another example, a human patient is diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 15 is administered to the patient with a pharmaceutically acceptable carrier. FIGURE 15 is a conjugate comprising an antibody construct, two targeting binding domains, and two immune stimulatory compounds. The antibody construct contains two scaffolds as shown in light gray and two scaffolds as shown in dark gray. The antibody construct comprises two antigen binding sites (1510 and 1515), and a portion of the two dark gray scaffolds contain Fc domains (1520 and 1545). The immune-stimulatory compounds (1530 and 1540) are conjugated to the antibody construct by linkers (1560 and 1570). The targeting binding domains are conjugated to the antibody construct (1580 and 1585).
EXAMPLE 23 Determination of Kd Values [0489] Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay.
[0490] Solution binding affinity of Fabs for antigen is measured by equilibrating the Fab with a minimal concentration of ( I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay, multi-well plates are coated overnight with 5 pg/mL of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 °C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 μΐ/well of scintillant is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
EXAMPLE 24
-248Determination of Kd Values [0491] Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 °C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/mL (~0.2 μΜ) before injection at a flow rate of 5 pL/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25 °C at a flow rate of approximately 25 pL/min. Association rates (kon) and dissociation rates (kOff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-l s-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25 °C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.
EXAMPLE 25 Lysine-based Bioconjugation [0296] The antibody construct is exchanged into an appropriate buffer, for example, phosphate, borate, PBS, Tris-Acetate at a concentration of about 2 mg/mL to about 10 mg/mL. An appropriate number of equivalents of the immune stimulatory compound-linker construct (ATAC) were added as a solution with stirring. Dependent on the physical properties of the immune stimulatory compoundlinker construct, a co-solvent was introduced prior to the addition of the immune stimulatory compound-linker construct to facilitate solubility. The reaction was stirred at room temperature for 2 hours to about 12 hours depending on the observed reactivity. The progression of the reaction was monitored by LC-MS. Once the reaction was deemed complete, the remaining immune stimulatory compound-linker constructs were removed by applicable methods and the antibody construct-249immune stimulatory compound conjugate was exchanged into the desired formulation buffer. Lysine-linked conjugates were synthesized starting with 10 mg of antibody (mAh) and 10 equivalents of ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, or ATAC10 using the conditions described in Scheme 34 below (ADC = antibody immune-stimulatory compound conjugate). Monomer content and immune-stimulatory compound-antibody construct ratios (molar ratios) were determined by methods described in EXAMPLES 27-31.
Scheme 34:
equivs of ATAC sodium phoshate mAb ___ADC pH = 8
20% v/v DMSO
Figure GB2552041A_D0299
Figure GB2552041A_D0300
TABLE 7
Scaffold Name Isolated ADC % Monomer DAR
i SBT-040-WT-ATAC4 9.7 mg > 95% 4.6
i SBT-040-G2-ATAC4 12.3 mg > 95% 2.9
ii SBT-040-WT-ATAC3 7.8 mg > 95% 5.4
ii SBT-040-G2-ATAC3 8.9 mg > 95% 3.3
ii SBT-040-WT-ATAC1 7.2 mg > 99% 1.9
ii SBT-040-G2-ATAC1 7.1 mg 99% 2.1
-250EXAMPLE 26
Cysteine-based Bioconjugation [0297] The antibody construct was exchanged into an appropriate buffer, for example, phosphate, borate, PBS, Tris-Acetate at a concentration of about 2 mg/mL to about 10 mg/mL with an appropriate number of equivalents of a reducing agent, for example, dithiothreitol or tris(2carboxyethyl)phosphine. The resultant solution was stirred for an appropriate amount of time and temperature to effect the desired reduction. The immune stimulatory compound-linker construct was added as a solution with stirring. Dependent on the physical properties of the immune stimulatory compound-linker construct, a co-solvent was introduced prior to the addition of the immune stimulatory compound-linker construct to facilitate solubility. The reaction was stirred at room temperature for about 1 hour to about 12 hours depending on the observed reactivity. The progression of the reaction was monitored by liquid chromatography-mass spectrometry (LC-MS). Once the reaction was deemed complete, the remaining free immune stimulatory compound-linker construct was removed by applicable methods and the antibody construct-immune stimulatory compound conjugate was exchanged into the desired formulation buffer. Such cysteine-based conjugates were synthesized starting with 10 mg of antibody (mAh) and 7 equivalents of ATAC11ATAC45 using the conditions described in Scheme 35 below (ADC = antibody immune-stimulatory compound conjugate). Monomer content and drug-antibody ratios can be determined by methods described in EXAMPLES 27-31.
Scheme 35:
1. reducing agent mAb ___ADC
2. 7 equivs of ATAC sodium phoshate pH = 8
20% v/v DMSO
-251OH
Figure GB2552041A_D0301
Figure GB2552041A_D0302
TABLE 8
Scaffold Name Isolated ADC % Monomer DAR
i SBT-040-WT-ATAC11 9.0 mg > 95% 4.5
i SBT-040-G2-ATAC11 8.9 mg 94% 4.0
ii SBT-040-WT-ATAC22 9.0 mg 93% 4.6
ii SBT-040-G2-ATAC22 9.0 mg > 95% 4.1
ii SBT-040-AAA-ATAC22 8.5 mg 98% 4.1
ii SBT-040-VLPLL-ATAC22 8.5 mg 94% 3.9
iii SBT-040-WT-ATAC12 11.7 mg > 95% 4.0
iii SBT-040-G2-ATAC12 11.4 mg 96% 4.1
iv SBT-040-WT-ATAC23 11.5 mg 93% 4.7
i SBT-040-AAA-ATAC11 10.2 mg > 95% 2.9
i SBT-040-VLPLL-ATAC11 9.4 mg 96% 2.9
iv SBT-040-AAA-ATAC23 7.1 mg > 95% 1.9
-252-
iv SBT-040-G1 (VLPLL)-ATAC23 7.7 mg > 95% 2.6
EXAMPLE 27
Determination of Molar Ratio [0298] This example illustrates one method by which the molar ratio is determined. One microgram of antibody construct immune-stimulatory compound conjugate is injected into an LC/MS such as an Agilent 6550 iFunnel Q-TOF equipped with an Agilent Dual Jet Stream ESI source coupled with Agilent 1290 Infinity UHPLC system. Raw data is obtained and is deconvoluted with software such as Agilent MassHunter Qualitative Analysis Software with BioConfirm using the Maximum Entropy deconvolution algorithm. The average mass of intact antibody construct immune-stimulatory compound conjugates is calculated by the software, which can use top peak height at 25% for the calculation. This data is then imported into another program to calculate the molar ratio of the antibody construct immune-stimulatory compound conjugate such as Agilent molar ratio calculator.
EXAMPLE 28
Determination of molar ratio for SBT-040-G1WT conjugated to a Cys-targeted compound [0492] FIGURE 28 shows HPLC analysis of SBT-040-G1WT conjugated to a Cys-targeted drug linker tool compound. First, 10 pL of a 5 mg/mL solution of the antibody-drug conjugate was injected into an HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography (HIC) column (2.5 μΜ particle size, 4.6 mm x 35 mm) attached. Then, over the course of 18 minutes, a method was run in which the mobile phase gradient ran from 100% mobile phase A to 100% mobile phase B over the course of 12 minutes, followed by a six minute reequilibration at 100% mobile phase A. The flow rate was 0.8 mL/min and the detector was set at 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the chromatogram was integrated and the molar ratio was determined by summing the weighted peak area. The molar ratio was calculated to be about 4.56 with 7% unconjugated antibody.
EXAMPLE 29
Determination of Molar Ratio for SBT-040-G1WT Conjugated to ATAC11 [0493] FIGURE 29 shows HPLC analysis of SBT-040-G1WT conjugated to ATAC11, which is a cleavable Maleimide-Val-Ala-PABA-Gardiquimod linker. First, 10 pL of a 5 mg/mL solution of the antibody immune-stimulatory compound conjugate was injected into an HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography (HIC) column (2.5 pM particle size, 4.6 mm x 35 mm) attached. Then, over the course of 18 minutes, a method was run in -253which the mobile phase gradient ran from 100% mobile phase A to 100% mobile phase B over the course of 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate was 0.8 mL/min and the detector was set at 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the chromatogram was integrated and the molar ratio was determined by summing the weighted peak area. The molar ratio was calculated to be about 4.5.
EXAMPLE 30
Determination of Molar Ratio for SBT-040-G2WT Conjugated to ATAC11 [0494] FIGURE 30 shows HPLC analysis of SBT-040-G2WT conjugated to ATAC11, which is a cleavable Maleimide-Val-Ala-PABA-Gardiquimod linker. First, 10 pL of a 5 mg/mL solution of the antibody-immune stimulatory compound conjugate was injected into an HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography (HIC) column (2.5 pM particle size, 4.6 mm x 35 mm) attached. Then, over the course of 18 minutes, a method was run in which the mobile phase gradient ran from 100% mobile phase A to 100% mobile phase B over the course of 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate was 0.8 mL/min and the detector was set at 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the chromatogram was integrated and the molar ratio was determined by summing the weighted peak area. The molar ratio was calculated to be about 4.0.
EXAMPLE 31
Additional Method for Determination of Molar Ratio [0495] Another method for determination of molar ratio is as follows. First, 10 pL of a 5 mg/mL solution of an antibody construct immune-stimulatory compound conjugate is injected into an HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography (HIC) column (2.5 pM particle size, 4.6 mm x 35 mm) attached. Then, over the course of 18 minutes, a method is run in which the mobile phase gradient is run from 100% mobile phase A to 100% mobile phase B over the course of 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate is 0.8 mL/min and the detector is set at 280 nM. Mobile phase A is 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B is 25% isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the chromatogram is integrated and the molar ratio is determined by summing the weighted peak area.
-254EXAMPLE 32
Affinity Measurement of Unconjugated Anti-CD40 Antibody or Anti-C40 Antibody ImmuneStimulatory Conjugate to Recombinant CD40 Ectodomain Fey Receptors Using BioLayer Interferometry [0496] This examples shows affinity measurements of unconjugated anti-C40 antibody or anti-CD40 antibody immune-stimulatory conjugate to Fc receptors (FcRs). Antibody affinity to its antigen (such as CD40) and the Fey Receptors (such as FcyRI, FcyRII, FcyRIII) was quantitated using BioLayer Interferometry (BLI). The anti-CD40 antibodies with different Fc (SBT-040-WT, SBT040-VLPLL, SBT-040-AAA and SBT-040-G2) were first produced as described in EXAMPLE 1 and then conjugated with an immune-stimulatory compound (any one of ATAC1 - ATAC34 and ATAC 43) as described in EXAMPLE 2. Following successful conjugation, their molecular interactions with CD40 Extracellular Domain (ECD) and various human FcyRs were quantitated using BLI.
[0497] Analysis of CD40 ECD interaction was performed using Octet Red 96 instrument (ForteBio). The Octet systems use propriety BLI to analyze biomolecular interaction. Unconjugated anti-CD40 antibodies (SBT-040-WT, SBT-040-VLPLL, SBT-040-AAA, SBT-040-G2) and anti-CD40 antibody immune-stimulatory compound conjugates were immobilized on anti-human Fc biosensors and incubated with varying concentration of monomeric human or rhesus CD40 ranging from 1.2 nM to 300 nM in PBS. The experiments were comprised of 5 steps: (1) baseline acquisition (60 s); (2) antibodies and antibody immune-stimulatory compound conjugates loading onto anti-human Fc biosensor (120 s); (3) second baseline acquisition (60 s); (4) association of interacting monomeric CD40 ECD protein for kon measurement (120 s); (5) Dissociation of interacting monomeric CD40 ECD for kOff measurement (360 s). The interacting monomeric CD40 ECD were used at 5-6 concentrations of 3-fold concentration series. Data were analyzed using Octet Data Analysis Sofware 9.0 (ForteBio) and fitted to the 1:1 binding model. Equilibrium dissociation constants (KD) were calculated by the ratio of kon to kOff. Selected data are shown in TABLE 9. All the anti-CD40 antibody immune-stimulatory compound conjugates had similar binding as unconjugated anti-CD40 antibody to monomeric human or rhesus CD40 ECD.
TABLE 9
Human CD40 ECD monomer Rhesus CD40 ECD monomer
Antibody/Conj ugate KD (nM) Ka(l/Ms) Kd (1/s) KD (nM) Ka(l/Ms) Kd (1/s)
SBT-040-WT 8.4 1.35E+5 1.13E-3 14.0 7.03E+4 9.87E-4
SBT-040-WT-ATAC11 8.3 1.24E+5 1.03E-3 13.5 7.56E+4 1.02E-3
-255-
SBT-040-WT-ATAC22 8.0 1.37E+5 1.09E-3 13.6 7.61E+4 1.04E-3
SBT-040-WT-ATAC4 9.3 1.04E+5 9.68E-4 12.9 6.55E+4 8.46E-4
SBT-040-WT-ATAC3 9.0 9.97E+5 8.97E-4 13.1 6.31E+4 8.23E-4
SBT-040-G2 7.7 1.34E+5 1.04E-3 12.4 7.61E+4 9.41E-4
SBT-040-G2-ATAC11 7.4 1.47E+5 1.09E-3 14.0 8.16E+4 1.14E-3
SBT-040-G2-ATAC22 7.2 1.42E+5 1.02E-3 15.2 7.20E+4 1.09E-3
SBT-040-G2-ATAC4 8.7 9.97E+5 8.67E-4 13.6 5.80E+4 7.88E-4
SBT-040-G2-ATAC3 7.3 1.15E+5 8.97E-4 11.5 7.21E+4 8.33E-4
SBT040-VLPLL 5.6 9.37E+4 5.26E-4 Not done
SBT040-VLPLL ATAC 11 7.6 9.75E+4 7.37E-4 Not done
SBT040-VLPLL ATAC22 7.5 1.02E+5 7.71E-4 Not done
SBT040-AAA 6.0 9.89E+4 5.97E-4 Not done
SBT040-AAA ATAC 11 4.7 1.61E+5 7.53E-4 Not done
SBT040-AAA ATAC22 5.0 1.36E+5 6.81E-4 Not done
[0498] Human Fey R interaction analysis was also performed using Octet Red 96 instrument. For human FcyRI and FcyRIIA interactions, unconjugated anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory compound conjugates were immobilized on anti-human Fc biosensors and incubated with varying concentration of monomeric FcyR ranging from 1.2 nM to 1 μΜ in PBS.
The experiments were comprised of 5 steps: (1) baseline acquisition (60 s); (2) anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory compound conjugates loading onto anti-human Fc biosensor (120 s); (3) second baseline acquisition (60 s); (4) association of interacting protein for kon measurement (120 s); (5) dissociation of interacting FcyR for kOff measurement (300 s). The interacting monomeric FcyR were used at 5-6 concentrations of 3-fold concentration series. Data were analyzed using Octet Data Analysis Software 9.0 (ForteBio) and fitted to the 1:1 binding model. Equilibrium dissociation constants (KD) were calculated by the ratio of kon to kOff. Selected data are shown in TABLE 10. There is very little to no changes in anti-CD40 antibody immune-stimulatory compound conjugate interaction with human FcyRI and FcyRIIA as compared to unconjugated antiCD40 antibody interactions with the respective FcyR monomeric protein.
[0499] For human FcyRIIB/C, FcyRIIIA F158, FcyRIIIA V158 and FcyRIIIB interaction studies, the proteins were immobilized on anti-his tag biosensors and incubated with varying concentration of unconjugated anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory compound
-256conjugates ranging from 0.04 μΜ to 8 μΜ. This format was chosen because of weak interactions if antibodies were captured first and FcyR added afterwards. The experiment comprised of 5 steps: (1) baseline acquisition (60 s); (2) anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory compound conjugates loading onto anti-human Fc biosensor (120 s); (3) second baseline acquisition (60 s); (4) association of interacting protein for kon measurement (120 s); (5) dissociation of interacting FcyR for kOff measurement (300 s). The interacting anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory compound conjugates were used at 4 concentrations of 2 fold concentration series. Data were analyzed using Octet Data Analysis Software 9.0 (ForteBio) and \ fitted to the avidity binding model. Equilibrium dissociation constants (KD) were calculated by the ratio of kon to kOff. Selected data are shown in TABLE 10. In most cases, there were no changes with the antibody immune-stimulatory compound conjugates with human FcyRIIB/C, FcyRIIIA FI58, FcyRIIIA VI58 and FcyRIIIB when compared to the parental unconjugated antibody. In some cases, there were small changes usually within 2 fold such as SBT040-G2 ATAC 11 interaction with FcyRIIIA F158 when compared to the unconjugated SBT040-G2.
TABLE 10
Antibody/Conj ugate FcyRI KD (1-1) Fc/RIIA KD (1-1) Fc/RIIB/C KD (avidity) Fc/RIIIA F158 KD (avidity) Fc/RIIIA V158 KD (avidity) Fc/RIIIB KD (avidity)
SBT-040-WT 0.68 nM 27 nM 1.60 uM 0.86 uM 0.51 uM 3.60 uM
SBT-040-WT- ATAC11 0.79 nM 35 nM 2.20 uM 1.21 uM 0.62 uM 5.00 uM
SBT-040-WT- ATAC22 0.80 nM 30 nM 1.68 uM 0.82 uM 0.41 uM 2.71 uM
SBT-040-WT- ATAC4 0.63 nM 22 nM 1.83 uM 0.83 uM 0.46 uM 3.24 uM
SBT-040-WT- ATAC3 0.77 nM 22 nM 0.97 uM 0.57 uM 0.30 uM 1.50 uM
SBT-040-G2 No binding 22 nM 2.62 uM 6.50 uM 3.65 uM No binding
SBT-040-G2- ATAC11 No binding 23 nM 3.78 uM 10.6 uM 4.11 uM No binding
SBT-040-G2- ATAC22 No binding 22 nM 3.16uM 7.06 uM 3.57 uM No binding
-257-
SBT-040-G2- ATAC4 No binding 22 nM 3.49 uM 7.00 uM 3.84 uM No binding
SBT-040-G2- ATAC3 No binding 17 nM 1.85 uM 8.09 uM 4.86 uM 3.93 uM
SBT040-VLPLL 1.5 nM 16 nM 2.30 uM 0.16 uM 0.08 uM 1.73 uM
SBT040-VLPLL ATAC11 1.3 nM 24 nM 3.36 uM 0.16 uM 0.08 uM 2.34 uM
SBT040-VLPLL ATAC22 1.2 nM 22 nM 2.14uM 0.14uM 0.08 uM 1.65 uM
SBT040-AAA 0.7 nM 64 nM 4.84 uM 0.47 uM 0.27 uM 2.44 uM
SBT040-AAA ATAC11 0.5 nM 97 nM 3.77 uM 0.59 uM 0.29 uM 3.83 uM
SBT040-AAA ATAC22 0.5 nM 116 nM 2.93 uM 0.45 uM 0.25 uM 2.56 uM
EXAMPLE 33
Stability of Anti-C40 Antibody Immune-Stimulatory Conjugates in IgG Depleted Human Serum [0500] Stability of the anti-CD40 antibody immune-stimulatory conjugates in human serum (IgG depleted) were measured over 96 hours at 37 °C using either a direct HIC-UV analysis approach (Method A) or an affinity capture approach (Method B). SBT-040-G1-ATAC12, SBT-040-G2ATAC12, or SBT-040-G1-ATAC30 were spiked in IgG-depleted human serum (BBI solutions #
SF142-2) in sterile tubes (75% final serum concentration) and samples were split into 4 aliquots of equal size then transferred to a 37 °C incubator. One of the aliquots of each sample was taken from the incubator at each time-point (T = Oh, 24h, 48h, 96h) and average drug-antibody ratios (DAR) were recorded.
Method A: Direct HIC-UV analysis [0501] At 0, 24, 48 and 96 hours after the beginning of incubation, the anti-CD40 antibody immunestimulatory conjugates spiked in IgG depleted human serum were analyzed by analytical hydrophobic interaction chromatography (HIC) using a TOSOH TSKgel Butyl-NPR 4.6 mm x 35 mm HIC column (TOSOH Bioscience, # 14947) connected to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hemstead, UK). Results are reported below in TABLE 11.
TABLE 11
Time (h) Average DAR
SBT-040-G1- SBT-040-G2- SBT-040-G1-
-258-
ATAC12 ATAC12 ATAC30
0 4.3 3.5 3.7
24 4.1 3.5 3.6
48 3.9 3.5 3.4
96 3.8 3.4 3.0
Method B: Affinity capture, de-glycosylation and RP-ESI-MS analysis [0502] ADCs were immunocaptured from the IgG depleted human serum using an anti-Human IgG (Fc specific) biotin antibody immobilized on streptavidin beads at 0, 24, 48 and 96 hours after the beginning of incubation. After elution from the beads, the samples were de-glycosylated using agarose-immobilized EndoS (Genovis Inc, USA). The de-glycosylated ADCs were analyzed by reverse phase chromatography hyphenated to electrospray ionization mass spectrometry (RP-ESIMS) using an Acquity nano UPLC in line with a Xevo G2S Q-TOF (Waters, Elstree, UK). The separation was performed using an Acquity UPLC online coupled to an ESI-MS mass spectrometer. Mass spectrometric analysis was performed in positive ion mode, scanning from 1000 to 4000 m/z in high mass operating mode. The ion envelope produced by each sample was deconvoluted using the MaxEntl algorithm provided within the MassLynx software (Waters, Elstree, UK). Results are reported in the table below.
Time (h) Average DAR
SBT-040-G1-ATAC4 SBT-040-G1-ATAC3
0 4.4 3.8
24 4.7 3.8
48 4.6 3.7
96 4.5 3.7
EXAMPLE 34 Synthesis of ATAC 18 [0503] This example shows the synthesis of (lR,6R,8R,9S,10S,15R,17R,18S)-8,17-Bis(2-amino-6oxo-1,9-dihy dropurin-9-yl)-18-(3 - { 2- [2-(2- { 2- [3-(2,5 -di oxo-1 H-pyrrol-1 yl)propionylamino]ethoxy}ethoxy)-ethoxy]ethoxy}propionylamino)-9-hydroxy-3,12-dioxy2.4.7.11.13.16-hexaoxa-3 λ5.12λ5 -diphosphatricy clo[ 13.3.0.06,10] octadecane-3,12-dione, triethylammonium salt (ATAC 18).
-259-
Figure GB2552041A_D0303
Figure GB2552041A_D0304
ATAC18
Step A: Preparation of Int ATAC 18-1 o
Figure GB2552041A_D0305
O lntATAC18-1 [0504] To a solution containing 100 mg (0.106 mmol) of Compound 21 in 5 mL of DMSO and 39 mg (0.106 mmol) of 3-[2-(2-{2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy}ethoxy)ethoxy] propionic acid was added 22 mg (0.16 mmol) of HOBT and 27 mg (0.212 mmol) of DIC. The reaction mixture was stirred at room temperature for 16h then purified by reverse phase chromatography without work-up. The resulting product fractions were lyophilized to afford 70 mg of product which was covered with 7 mL of a 1:2 mixture of TFA and CH2CI2. The mixture was stirred for 2h at ambient temperature before the solvent was removed. The resulting residue Int ATAC18-1 was used directly in the next step without purification.
Step B: Preparation of Int ATAC 18-2
-260ο
Figure GB2552041A_D0306
ο 2 CH3NH3+
Int ATAC18-2 [0505] A solution containing Int ATAC18-1 (50 mg, 0.04 mmol) and 3.0 mL of methylamine (33% in anhydrous ethanol) was stirred for 16h at room temperature. The reaction mixture was concentrated to provide Int ATAC18-2 as a white foam which used directly in the next step. LCMS (ESI, m/z)·. 1051 [M+H],
Step C: Preparation oflntATAC 18-3 o
Figure GB2552041A_D0307
ATAC18-3 [0506] The above crude Int ATAC18-2 was azeotroped with 3:1 pyridine/triethylamine three times then dissolved in 0.8 mL pyridine. To this solution at 55°C was added 2 mL triethylamine and ImL triethylamine trihydrofluride simultaneously. After stirring lh, the bath was removed and anhydrous acetone was added immediately. The mixture was stirred for 20 min and the white solid was collected by filtration. The precipitate was washed with anhydrous acetone. The product was
-261purified by preparative flash chromatography. The resulting solution was lyophilized to provide 3 mg of Int ATAC18-3 as a white solid. LCMS: (ESI, m/z): 937.6 [M+H]+. 'H NMR (DMSO-d6): δ
7.99 (s, 1H), 7.89 (s, 1H), 5.73 (m, 2H), 5.06 (m, 1H); 4.84-4.74 (m, 2H); 4.53 (t, J=5.1 Hz, 1H),
4.21 (m, 1H), 2.96 (m, 2H), 3.73-3.29 (m, 16H), 2.87 (q, J=7.2 Hz, 8H), 2.35 (m, 2H), 1.10 (t, J=7.2
Hz, 12H). 31P-NMR (DMSO-d6) δ 0.25, -1.32.
Step D: Preparation of ATAC 18 r
HN
Figure GB2552041A_D0308
ATAC18 [0507] To a solution containing 2.0 mg (0.002 mmol) of Int ATAC18-3 in 0.2 mL of DCM was added 0.5 mg (0.004 mmol) of DIPEA then 1.06 mg (0.004 mmol) of 2,5-dioxo-l-pyrrolidinyl 3(2,5-dioxo-lH-pyrrol-l-yl)propionate. The resulting solution was stirred overnight then the solvent was evaporated. Reverse phase column chromatography afforded the desired compound ATAC18 as a white solid. LCMS: (ESI, m/z): 1088 [M+H]+. 'H-NMR (D2O) δ 8.15 (s, 1H), 8.05 (s, 1H), 7.07 (s, 2H), 5.88-5.84 (m, 2H), 5.26 (m, 1H), 5.05-4.91 (m, 2H), 4.76 (m, 1H), 4.35 (m, 1H), 4.29-4.20 (m, 5H), 3.77 (t, 2H), 3.7-3.5 (m, 16H), 3.33 (m, 2H), 3.15 (q, J=7.2 Hz, 15H), 2.52 (m, 1H), 1.25 (t, J=7.2 Hz, 21H). 31P-NMR (DMSO-d6) δ 1.51, 0.62.
EXAMPLE 35 Synthesis of ATAC 43 [0508] This example shows the synthesis of 2,3,4,5,6-Pentafluorophenyl 5{(1 S,6R,8R,9S, 10R, 15R, 17R, 18 S)-8,17-bis(2-amino-6-oxo-l,9-dihydropurin-9-yl)-18-hydroxy-2623,12-dioxo-3,12-dioxy-2.4.7.11.13.16-hexaoxa-3k5.12Z5-diphosphatricyclo[ 13.3.0.06,10]octadec-9ylamino}-5-oxovalerate triethyl ammonium salt (ATAC 43)
Figure GB2552041A_D0309
Step A: Preparation of Int ATAC 43-1 o
Figure GB2552041A_D0310
Compound 2
Ο
Figure GB2552041A_D0311
Int ATAC 43-1 [0509] To a solution containing 50 mg (0.072 mmol) of Compound 2 in 2.5 mL of DMSO was added 57 mg (0.72 mmol) of pyridine and 5 mg of DMAP. The resulting solution was stirred for 10 minutes then treated with 82 mg (0.72 mmol) of glutaric anhydride. The reaction mixture was stirred at room temperature for 16h then purified by reverse phase chromatography without work-up. Fractions containing product were lyophilized over 48h to provide 10 mg of Int ATAC 43-1 as a white solid. LCMS (ESI, m/z)·. 804 [M+H], 'H NMR (DMSO-d6) δ 10.6 (bs, 2H), 9.20 (bs, 1H),
7.99 (s, 1H), 7.92 (s, 1H), 7.56 (bs, 1H), 6.56 (m, 4H), 5.76 (dd, J=8.1, 17.7 Hz, 2H), 4.94 (m, 1H),
4.76-4.68 (m, 2H), 4.58 (m, 1H), 4.22 (m, 1H), 4.03-3.91 (m, 6H), 3.97 (q, J=7.0Hz, 12H), 2.21 (t,
J=7.2Hz, 2H), 2.09-1.99 m, 2H), 1.67-1.60 (m, 2H), 1.20 (t, J=7.2Hz, 18H). 31P NMR (DMSO-d6) d
0.44, -1.3.
Step B: Preparation of ATAC43
-263-
Figure GB2552041A_D0312
Int ATAC 43-1 ATAC 43 [0510] To a solution containing 7.0 mg (0.009 mmol) of Int ATAC 43-1 and 2.6 mg (0.013 mmol, 1.5 eq) of pentafluorophenol in 300 uL of DMSO was added 1.8 mg (0.012 mmol, 1.3 eq) of EDC and the reaction mixture was stirred for 16h at room temperature then purified by reverse phase chromatography without work-up. Fractions containing product were lyophilized to provide ATAC 43 as a white solid. LCMS (ESI, m/z)·. 970 [M+H],
EXAMPLE 36
STING Agonist Screening Assay [0511] Biology materials and general procedures. The following reporter cell lines, reagents and ligands were obtained from InvivoGen: THP-1 Dual cells (thpd-nfis); THP-1 Dual KO-STING cells (thpd-kostg); Quanti-Blue (rep-qbl); Quanti-Luc (rep-qlcl); Normocin (ant-nr-1); Zeocin (ant-zn-1); Blasticidin (ant-bl-1); PMA (tlrl-pma); 2’,3’-cGAMP (tlrl-nacga); and 2’3’-c-di-AM(PS)2 (Rp,Rp) (tlrl-nacda2r). 3’,3’-cGAMP (SML 1232) and 3’,5’-cyclic-di-GMP (SML 1228) were purchased from Sigma. THP-1 Dual cells were cultured in RPMI 1640 (Lonza) supplemented with 10% fetal bovine serum, 2mM glutamine, 50 pg/mL penicillin, 50 U/mL streptomycin (all from Gibco). Cells were passaged at 0.7 x 106 cell/mL every 2-3 days. 100 pg/mL Normocin, 100 pg/mL Zeocin, and 10 pg/mL Blasticidin were added every other passage to maintain reporter expression according to the manufacturer’s instructions.
[0512] General procedure for in vitro screening of CDNs for cytokine induction activity. THP1 Dual or THP-1 Dual KO-STING cells were plated at 50,000 cells per well in 200 pL of culture media in 96-well plates and matured with 150 nM PMA for 16-18 hours. Cells were washed in culture media the following day and supernatants were removed. Cells were stimulated for 30 min at 37°C in a 5% CO2 incubator with 150 pL of CDNs prepared in permeabilization buffer with 2 pg/mL digitonin (CalBiochem) at four different concentrations (1, 0.1, 0.01 and 0.001 pM). After the incubation, the permeabilization buffer and CDNs were removed and replaced with 150 pL culture media. Cells were incubated an additional 23.5 h at 37°C in a 5% CO2 incubator. Prior to
-264supernatnant harvest, cells were spun at 300 x g for 5 min to remove cell debris. ISG54 activity was indirectly quantified from supernatants using QUANTI-Luc, which was prepared and used according to the manufacturer’s instructions. NF-κΒ secreted alkaline phosphatase pathway activity was indirectly quantified from supernatants using QUANTI-Blue, which was prepared and used according to the manufacturer’s instructions. CellTiterGlo (Promega) was used to lyse and assess viability according to the manufacturer’s instructions. Plates were analyzed on Envision (Perkin Elmer) or Synergy (BioTek) plate readers.
EXAMPLE 37
Tumor Size in a Mouse Tumor Model Is Reduced by Anti-C40 Antibody Immune-Stimulatory Compound Conjugates [0513] The example shows tumor size is reduced in a mouse tumor model after administration of an anti-CD40 antibody immune-stimulatory compound conjugate. The immunocompromised mouse strain NSG (NOD.Cg-PrkdcscldIL-2rgtmlW'il/SzJ) into which human tumor cells are co-injected with human T cells and myeloid dendritic cells (mDC) is used as the mouse tumor model. In vivo immune-mediated activity of the anti-CD40 antibody immune-stimulatory compound conjugate is assessed by examining human mDCs in this model.
[0514] Anti-CD40 antibody immune-stimulatory compound conjugates or anti-CD40 antibody (control) are injected intraperitoneally to mice. Immediately after this injection, Raji cells (a B cell lymphoma tumor cell line), human T cells, and mDCs from the same human donor are co-injected into the mice. The Raji cells are injected subcutaneously at a concentration of 1χ10Λ7 cells/mouse. The human T cells are injected at a concentration of 1χ10Λ6 cells/mouse, and mDCs are injected at a concentration of 5χ10Λ5 cells/mouse. Tumor growth is measured using calipers twice per week, beginning seven days post tumor cell transfer and ending at study termination, approximately 3 weeks after tumor cell inoculation. Tumor size is reduced in mice that received the anti-CD40 antibody immune-stimulatory compound conjugates in comparison to mice that received the antiCD40 antibody.
EXAMPLE 38
Dendritic Cell Priming of T Cells Is Enhanced by Anti-C40 Antibody Immune-Stimulatory
Compound Conjugates [0515] This example shows that dendritic cell priming of T cells is enhanced by anti-CD40 antibody immune-stimulatory compound conjugates. After anti-CD40 antibody immune-stimulatory compound conjugates is administered, the upregulation of co-stimulatory molecules and cytokine
-265production by dendritic cells is induced. Priming of a T cell response is therefore enhanced by the stimulation of dendritic cells in this manner. An in vitro human mDC and T cell co-culture assay is used to demonstrate this.
[0516] Myeloid dendritic cells (mDCs) and allogeneic T cells are isolated from peripheral blood mononuclear cells (PBMCs), are labeled with a dye to monitor cell division, and are co-cultured for 5 days in the presence of the anti-CD40 antibody immune-stimulatory compound conjugates or an isotype control. T cell activation is assessed as percent dividing cells as measured by flow cytometry using the indicator dye. The percent of dividing T cells is increased in T cells co-cultured with the anti-CD40 antibody immune-stimulatory compound conjugates in comparison to the percent of dividing T cells for T cells co-cultured with the isotype control.
[0517] While aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the aspects of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
-266-

Claims (139)

  1. WHAT IS CLAIMED IS:
    1. A conjugate comprising:
    a) an immune-stimulatory compound;
    b) an antibody construct comprising an antigen binding domain and an Fc domain, wherein said antigen binding domain binds to a first antigen and wherein a Ka for binding of said Fc domain to an Fc receptor in the presence of said immune-stimulatory compound is no greater than about 100 times a Ka for binding of said Fc domain to said Fc receptor in the absence of the immune stimulatory compound; and
    c) a linker, wherein said linker attaches said antibody construct to said immunestimulatory compound.
  2. 2. The conjugate of claim 1, wherein said antigen binding domain binds said first antigen in a presence of said immune-stimulatory compound.
  3. 3. The conjugate of any of claims 1-2, wherein a Kafor binding of said antigen binding domain to said first antigen in a presence of said immune-stimulatory compound is less than about 100 nM and no greater than about 100 times a Ka for binding of said antigen binding domain to said first antigen in the absence of said immune-stimulatory compound.
  4. 4. The conjugate of any of claims 1-2, wherein said Ka for binding of said antigen binding domain to said first antigen in the presence of said immune-stimulatory compound is less than about lOOnM and is no greater than about 10 times the Ka of the binding of the antigen binding domain to said first antigen in the absence of the immune-stimulatory compound; and said Ka for binding of said Fc domain to said Fc receptor in the presence of said immune-stimulatory compound is no greater than about 10 times said Ka for the binding of said Fc domain to said Fc receptor in the absence of said immune stimulatory compound.
  5. 5. The conjugate of any of claims 1-4, wherein a molar ratio of immune-stimulatory compound to antibody construct is less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2.
  6. 6. The conjugate of any one of claims 1-5 further comprising a targeting binding domain, wherein said targeting domain is attached to said antibody construct.
  7. 7. The conjugate of claim 6, wherein said targeting binding domain binds a second antigen.
  8. 8. The conjugate of any one of claims 6-7, wherein said targeting binding domain is attached to said antibody construct at a C-terminal end of said Fc domain.
    -2679. The conjugate of any of claims 1-8, wherein said antigen binding domain is from an antibody or non-antibody scaffold.
  9. 10. The conjugate of any of claims 1-9, wherein said antigen binding domain is at least 80% homologous to an antigen binding domain from an antibody or non-antibody scaffold.
  10. 11. The conjugate of any of claims 9-10, wherein said non-antibody scaffold is a DARPin, affimer, avimer, knottin, monobody, or affinity clamp.
  11. 12. The conjugate of any of claims 9-11, wherein said antigen binding domain is at least 80% homologous to an antigen binding domain from a DARPin, affimer, avimer, knottin, monobody, or affinity clamp.
  12. 13. The conjugate of any of claims 1-12, wherein said antigen binding domain recognizes a single antigen.
  13. 14. The conjugate of any of claims 1-13, wherein said antigen binding domain recognizes two or more antigens.
  14. 15. The conjugate of any of claims 1-14, wherein said first antigen is a tumor antigen.
  15. 16. The conjugate of any of claims 1-15, wherein said first antigen that is at least 80% homologous to CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAAI, MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Le\ CA-125, CA19-9, epidermal growth factor, pl85HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA/MARTl, Ras mutant, gplOO, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OYTES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL 1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC15, MSLN, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, or Fos-related antigen 1.
  16. 17. The conjugate of any of claims 1-16, wherein said first antigen is expressed on an immune cell.
  17. 18. The conjugate of any of claims 1-17, wherein said first antigen is expressed on an antigenpresenting cell.
    -26819. The conjugate of claim 1-18, wherein said first antigen is expressed on a dendritic cell, a macrophage, or a B-cell.
  18. 20. The conjugate of any of claims 1-19, wherein said first antigen is CD40.
  19. 21. The conjugate of any of claims 1-20, wherein said antigen binding domain is a CD40 agonist.
  20. 22. The conjugate of any of claims 1-21, wherein said antibody construct is an antibody.
  21. 23. The conjugate of any of claims 1-22, wherein said antibody construct is a human antibody or a humanized antibody.
  22. 24. The conjugate of any of claims 1-23, wherein said antibody construct comprises a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 4, at least 80%, 90%, or 100% homologous to SEQ ID NO: 26, or at least 80%, 90%, or 100% homologous to SEQ ID NO: 34.
  23. 25. The conjugate of any of claims 1-24, wherein said antibody construct comprises a light chain variable domain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 6.
  24. 26. The conjugate of any of claims 1-25, wherein said antibody construct comprises:
    a) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 15;
    b) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 16;
    c) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 17;
    d) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 18;
    e) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 22; or
    f) heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 30.
  25. 27. The conjugate of any of claims 1-26, wherein said antibody construct comprises a heavy chain variable domain that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 20.
  26. 28. The conjugate of any of claims 1-27, wherein said antibody binding domain comprises at least 80%, 90%, or 100% homology to:
    a) HC CDR1 comprising an amino acid sequence of SEQ ID NO: 23, HC CDR2 comprising an amino acid sequence of SEQ ID NO: 24, a HC CDR3 comprising an amino acid sequence of SEQ ID NO: 25, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 27, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 28, and LC CDR3 comprising an amino acid sequence of SEQ ID NO: 29; or
    b) HC CDR1 comprising an amino acid sequence of SEQ ID NO: 31, HC CDR2 comprising an amino acid sequence of SEQ ID NO: 32, a HC CDR3 comprising an amino acid sequence of SEQ ID NO: 33, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 35, LC CDR1
    -269comprising an amino acid sequence of SEQ ID NO: 36, and LC CDR3 comprising an amino acid sequence of SEQ ID NO: 37.
  27. 29. The conjugate of any of claims 1-28, wherein said Fc domain is from an antibody.
  28. 30. The conjugate of any of claims 1-29, wherein said Fc domain is at least 80% homologous to an Fc domain from an antibody.
  29. 31. The conjugate of any of claims 1-30, wherein said Fc domain binding to said Fc receptor in the presence of said immune-stimulatory compound results in Fc-receptor-mediated signaling.
  30. 32. The conjugate of any of claims 1-31, wherein said Fc domain binding to said Fc receptor in the presence of said immune-stimulatory compound results increased antigen presentation on an immune cell.
  31. 33. The conjugate of any of claims 1-32, wherein said Fc domain is a human Fc domain.
  32. 34. The conjugate of any of claims 1-33, wherein said Fc domain is selected from a group consisting of a human IgGl Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 Fc domain.
  33. 35. The conjugate of any of claims 1-34, wherein said Fc domain is an Fc domain variant comprising at least one amino acid residue change as compared to a wild type sequence of said Fc domain.
  34. 36. The conjugate of claims 1-35, wherein said Fc domain binds said Fc receptor with altered affinity as compared to a wild type Fc domain.
  35. 37. The conjugate of any one claims 1-36, wherein said Fc receptor is selected from a group consisting of CD16a, CD16b, CD32a, CD32b, and CD64.
  36. 38. The conjugate of any one of claims 1-37, wherein said Fc receptor is a CD16a F158 variant or a CD 16a V158 variant.
  37. 39. The conjugate of any one of claims 1-38, wherein said Fc domain binds said Fc receptor with higher affinity than a wild type Fc domain.
  38. 40. The conjugate of any one of claims 1-39, wherein said Fc receptor is selected from a group consisting of CD 16a, CD 16b, CD32a, CD32b, or CD64.
  39. 41. The conjugate of any one of claims 1-40, wherein said Fc receptor is a CD16a F158 variant or a CD 16a V158 variant.
  40. 42. The conjugate of any one of claims 1-41, wherein said Fc domain has at least one amino acid residue change as compared to wildtype, wherein said at least one amino acid residue change is:
    a) F243L, R292P, Y300L, L235V, and P396L, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Rabat;
    -270b) S239D and I332E, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Kabat; or
    c) S298A, E333A, and K334A, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Kabat.
  41. 43. The conjugate of any of claims 1-42, wherein said immune-stimulatory compound is a damageassociated molecular pattern molecule or a pathogen associated molecular pattern molecule.
  42. 44. The conjugate of any of claims 1-43, wherein said immune-stimulatory compound is a toll-like receptor agonist, STING agonist, or RIG-I agonist.
  43. 45. The conjugate of any of claims 1-44, wherein said immune-stimulatory compound is a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, or a TLR 10 agonist
  44. 46. The conjugate of any of claims 1-45, wherein said immune-stimulatory compound is selected from a group consisting of: S-27609, CL307, UC-IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS-1463GS-9620, GSK2245035, TMX-101, TMX-201, TMX-202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B, and CL663.
  45. 47. The conjugate of any of claims 1-46, wherein said immune-stimulatory compound comprises one or more rings selected from carbocyclic and heterocyclic rings.
  46. 48. The conjugate of any of claims 1-47, wherein said linker is covalently attached to said antibody construct.
  47. 49. The conjugated of any of claims 1-48, wherein said linker is covalently attached to said immunestimulatory compound.
  48. 50. The conjugate of any of claims 1-49, wherein said linker is not attached to an amino acid residue of said Fc domain selected from a group consisting of: 221, 222, 224, 227, 228, 230, 231, 223,
    233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 258, 262, 263,
    264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 283, 285, 286,
    288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305, 313, 317, 318, 320, 322,
    323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 336, 396, or 428, wherein numbering of amino acid residues in said Fc domain is according to the EU index as in Kabat.
  49. 51. The conjugate of any of claims 1-50, wherein said linker is attached to an amino acid residue of said antibody construct by a THIOMAB linker, or a Sortase A-catalyzed linker.
  50. 52. The conjugate of any of claims 1-51, wherein said linker is attached to said antibody construct via a sulfhydryl group, a primary amine, a hinge cysteine, a Cl lysine, an engineered cysteine in
    -271a light chain, an engineered light chain glutamine, or an unnatural amino acid engineered into a light chain or heavy chain.
  51. 53. The conjugate of any of claims 1-52, wherein said linker does not interfere with said Fc domain binding to said Fc receptor when said linker is attached to said antibody construct at an amino acid residue.
  52. 54. The conjugate of any of claims 1-53, wherein said linker does not interfere with Fc-receptormediated signaling resulting from said Fc domain binding to said Fc receptor when said linker is attached to said Fc domain at an amino acid residue.
  53. 55. The conjugate any of claims 1-54, wherein said linker is attached to said immune-stimulatory compound via an exocyclic nitrogen or carbon atom of said immune-stimulatory compound.
  54. 56. The conjugate of any of claims 1-55, wherein said immune-stimulatory compound is covalently attached to said linker by a bond to an exocyclic carbon or nitrogen atom on said immunestimulatory compound.
  55. 57. The conjugate of any of claims 1-56, wherein said linker is a peptide.
  56. 58. The conjugate of any of claims 1-57, wherein said linker is a cleavable linker.
  57. 59. The conjugate of any of claims 1-58, wherein said linker selected from a group consisting of:
    a) a valine-citrulline linker;
    b) a valine-citrulline linker containing a pentafluorophenyl group;
    c) a valine-citrulline linker containing a succinimide group;
    d) a valine-citrulline linker containing a para aminobenzoic acid group;
    e) a valine-citrulline linker containing a para aminobenzoic acid group and a pentafluorophenyl group; and
    f) a valine-citrulline linker containing a para aminobenzoic acid group and a succinimide group.
  58. 60. The conjugate of any of claims 1-59, wherein said linker is a non-cleavable linker.
  59. 61. The conjugate of any of claims 1-60, wherein said linker selected from a group consisting of:
    a) a maleimidocaproyl linker;
    b) a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules;
    c) a maleimide-PEG4 linker;
    d) a maleimidocaproyl linker containing a succinimide group;
    e) a maleimidocaproyl linker containing a pentafluorophenyl group;
    -272f) a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules; and
    g) a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules.
  60. 62. The conjugate of any of claims 1-61, wherein said conjugate induces the secretion of cytokine by an antigen presenting cell.
  61. 63. The conjugate of claim 62, wherein said cytokine is IFN-γ, IL-8, IL-12, IL-2, or a combination thereof.
  62. 64. The conjugate of any of claims 1-63, wherein said conjugate induces antigen presentation on an antigen presenting cell.
  63. 65. The conjugate of any of claims 1-64, wherein said conjugate is formulated to treat tumors.
  64. 66. The conjugate of any of claims 1-65, wherein said conjugate is in a pharmaceutical formulation.
  65. 67. A pharmaceutical composition comprising said conjugate of any of claims 1-66 and a pharmaceutically acceptable carrier.
  66. 68. A method of producing the conjugate of any of claims 1-66, comprising:
    a) selecting an antibody construct;
    b) selecting an immune-stimulatory compound; and
    c) attaching said antibody construct to said immune-stimulatory compound, wherein said immune-stimulatory compound is attached to said antibody construct via a linker and said antibody construct comprises an antigen binding domain and an Fc domain, wherein said antigen binding domain specifically binds an antigen in the presence of said immunestimulatory compound and said Fc domain specifically binds an Fc receptor in the presence of said immune-stimulatory compound.
  67. 69. A method of producing the conjugate of any of claims 6-66, comprising:
    a) selecting an antibody construct;
    b) selecting an immune-stimulatory compound;
    c) selecting a targeting binding domain;
    d) attaching said targeting binding domain to said antibody construct; and
    e) attaching said antibody construct to said immune-stimulatory compound, wherein said immune-stimulatory compound is attached to said antibody construct via a linker, wherein said antigen binding domain specifically binds a first antigen in the presence of said immunestimulatory compound and said targeting binding specifically binds a second antigen in the presence of said immune-stimulatory compound.
    -273ΊΟ. A method for treating a subject in need thereof, comprising administering a therapeutic dose of said conjugate of any one of claims 1-66 or said pharmaceutical composition of claim 67.
  68. 71. The method of claim 70, wherein said subject has cancer.
  69. 72. The method of any of claims 70-71, wherein said composition is administered intravenously, cutaneously, subcutaneously, or injected at a site of affliction.
  70. 73. A kit comprising of said conjugate of any one of claims 1-66.
  71. 74. A composition comprising a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 4 and heavy chain sequence that is at least 80%, 90%, or 100%
  72. 75. A composition comprising:
    a) a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 4 or at least 80%, 90%, or 100% homologous to SEQ ID NO: 26; and
    b) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 16, at least 80%, 90%, or 100% homologous to SEQ ID NO: 17, or at least 80%, 90%, or 100% homologous to SEQ ID NO: 18.
  73. 76. The composition of claim 75, wherein said composition binds to an Fc receptor with greater affinity than an antibody comprising a heavy chain sequence of SEQ ID NO: 15 or SEQ ID NO:
    22.
    A compound represented by the structure of Formula (I):
    Y O ,0 N(R )2 (I) or pharmaceutically acceptable salts thereof, wherein:
    X1 is selected from -OR2 and -SR2;
    X2 is selected from -OR3 and -SR3;
    B and B are independently selected from optionally substituted nitrogenous bases;
    Y is selected from —OR4, -NR4R4, and halogen;
    R1, R2, R3 and R4 are independently selected at each occurrence from hydrogen, -C(=O)R100, C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, each of which is
    -274independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R , R , R and R is independently optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, -OC(O)R100, -no2, =0, =s, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci^ alkyl, C2.6 alkenyl, and C2.6 alkynyl; and R100 at each occurrence is independently selected from hydrogen; and Cmo alkyl, C2_io alkenyl, C2_io alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -N02, =0, =S, and haloalkyl.
  74. 78. The compound or salt of claim 77, wherein the compound of Formula (I) is represented by Formula (IA):
    (IA) or pharmaceutically acceptable salts thereof.
  75. 79. The compound of salt of claim 77, wherein the compound of Formula (I) is represented by Formula (IB):
    (IB) or a pharmaceutically acceptable salt thereof.
    -2751 2
  76. 80. The compound or salt of any one of claims 77-79, wherein B and B are independently selected from optionally substituted purines.
  77. 81. The compound or salt of claim 80, wherein B and B are independently selected from optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, and optionally substituted isoguanine.
  78. 82. The compound or salt of claim 81, wherein B and B are independently selected from optionally substituted adenine and optionally substituted guanine.
  79. 83. The compound or salt of any one of claims 77-82, wherein B and B are independently optionally substituted with one or more substituents, wherein the optional substituents on B1 and B2 are independently selected at each occurrence from halogen, =0, =S, -OR100, -SR100, N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, NO2, -P(O)(OR100)2, -OP(O)(ORw°)2 and -CN; Cmo alkyl, C2-io alkenyl, C24o alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle is independently optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci.6 alkyl, C2.6 alkenyl, and C2.6 alkynyl.
  80. 84. The compound or salt of claim 83, wherein B and B are independently optionally substituted with one or more substituents, wherein the optional substituents on B and B are independently selected at each occurrence from halogen, =0, =S, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -N02, -P(O)(OR100)2, -OP(O)(OR100)2, -CN and Cmo alkyl.
  81. 85. The compound or salt of any one of claims 77-84, wherein B1 is an optionally substituted guanine.
  82. 86. The compound or salt of any one of claims 77-85, wherein B is an optionally substituted guanine.
  83. 87. The compound or salt of any one of claims 77-86, wherein X1 is selected from -OH and -SH.
  84. 88. The compound or salt of claim 87, wherein is X1 is -OH.
  85. 89. The compound or salt of any one of claims 77-88, wherein X is selected from -OH and -SH.
    -27690. The compound or salt of claim 89, wherein is X is -OH.
  86. 91. The compound of any one of claims 77-90, wherein Y is selected from -OH, -Ο-Cmo alkyl, NH(Cmo alkyl), and -NH2.
  87. 92. The compound of claim 91, wherein Y is -OH.
  88. 93. The compound of any one of claims 77-92, wherein R100 is independently selected at each occurrence from hydrogen and Cmo alkyl optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO2, =0, and =S.
  89. 94. The compound or salt of any one of claims 77-93, wherein the compound of Formula (I) is represented by Formula (IC):
    or a pharmaceutically acceptable salt thereof.
  90. 95. The compound or salt of claim 94, wherein the compound of Formula (IC) is represented by Formula (ID):
    or a pharmaceutically acceptable salt thereof.
  91. 96. The compound or salt of any one of claims 77-95, wherein the compound is a pharmaceutically acceptable salt.
  92. 97. The compound or salt of any one of the claims 77-96, wherein the compound or salt agonizes a stimulator of interferon genes (STING).
  93. 98. An antibody construct immune-stimulatory compound conjugate, comprising a compound or salt of any one of claims 77-97, an antibody, and a linker group, wherein the compound or salt is linked to the antibody through the linker group.
    -27799. The antibody construct immune-stimulatory compound conjugate of claim 98, wherein the linker group is selected from a cleavable or non-cleavable linker.
  94. 100. A compound represented by the structure of Formula (II):
    or pharmaceutically acceptable salts thereof, wherein:
    X1 is selected from -OR2 and -SR2;
    X2 is selected from -OR3 and -SR3;
    B and B are independently selected from optionally substituted nitrogenous bases, wherein each optional substituent is independently selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, R6, and -X3;
    Y is selected from —OR4, -SR4,-NR4R4, and halogen;
    Z is selected from —OR5, -SR5, and -NR5R5;
    R1, R2, R3, R4, and R5 are independently selected from a -X3; hydrogen, -C(=O)R100, -C(=O)OR100 and -C(=O)NR100; Ci -io alkyl, C2-io alkenyl, C2-io alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, N(R100)2, -S(O)R100, -S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R , R , R , R , and R is optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100 C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci_6 alkyl, C2.6 alkenyl, C2.6 alkynyl;
    R6 is independently selected from -C(=O)R100, -C(=O)OR100 and -C(=O)NR100; Cmo alkyl, C2_io alkenyl, C2_io alkynyl, each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, S(O)2R100, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S,
    -278=N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, C3-10 carbocycle and 3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in R6 is optionally substituted with one or more substituents selected from halogen, -OR100, -SR100, -N(R100)2, -S(O)R100, -S(O)2R100-C(O)R100, -C(O)OR100, OC(O)R100, -NO2, =0, =S, =N(R100), -P(O)(OR100)2, -OP(O)(OR100)2, -CN, Ci^ alkyl, C2.6 alkenyl, C2-6 alkynyl;
    R100 at each occurrence is independently selected from hydrogen; and Cmo alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle each of which is independently optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO2, =0, =S, and haloalkyl; and
    3 12345121 2
    X is a linker moiety, wherein at least one of R ,R,R,R,R,X,X,aB substituent and a B β
    substituent is -X .
  95. 101. The compound or salt of claim 100, wherein the compound of Formula (II) is represented by a structure of Formula (IIA):
    or pharmaceutically acceptable salts thereof.
  96. 102. The compound or salt of claim 100, wherein the compound of Formula (II) is represented by a structure of Formula (IIB):
    (IIB) or a pharmaceutically acceptable salt thereof.
    -2791 2
  97. 103. The compound of salt of any one of claims 100-102, wherein B and B are independently selected from optionally substituted purines.
  98. 104. The compound or salt of claim 103, wherein B and B are each, independently selected from one another, adenine, guanine, and derivatives thereof.
  99. 105. The compound or salt of claim 104, wherein B and B are independently selected from optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid, and optionally substituted isoguanine.
  100. 106. The compound or salt of claim 105, wherein B and B are independently selected from optionally substituted adenine and optionally substituted guanine.
  101. 107. The compound or salt of any one of claims 100-106, wherein B is substituted by X and optionally one or more additional substituents independently selected from halogen, -OR100, SR100, -N(R100)2, -S(O)R100, -S(0)2Rwo, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, and R6.
  102. 108. The compound or salt of claim 107, wherein B1 is represented by:
    /X2 3 *
    HN ιΛ/W ?
    and wherein B1 is optionally further substituted by one or more substituents.
  103. 109. The compound or salt of any one of claims 100 to 108, wherein B is substituted by X and optionally one or more additional substituents independently selected from halogen, -OR100, SR100, -N(R100)2, -S(O)R100, -S(0)2Rwo, -C(O)R100, -C(O)OR100, -OC(O)R100, -NO2, =0, =S, =N(R100), -CN, and R6.
  104. 110. The compound or salt of claim 109, wherein B is represented by:
    and wherein B is optionally further substituted by one or more substituents.
  105. 111. The compound or salt of any one of claims 100-108, wherein X is selected from -Ο- X and-S-X3.
    -280112. The compound or salt of any one of claims 100-111, wherein X1 is selected from -OH and SH.
  106. 113. The compound or salt of any one of claims 100-108, wherein X is selected from -Ο- X and-S-X3.
  107. 114. The compound or salt of any one of claims 100-113, wherein X is selected from -OH and SH.
  108. 115. The compound or salt of any one of claims 100-108, wherein Y is selected from -NR X , -SX3, and -Ο- X3.
  109. 116. The compound or salt of any one of claims 100-115, wherein Y is selected from -OH, -SH, O-Ci-io alkyl, -NH(Cmo alkyl), and -NH2
  110. 117. The compound or salt of any one of claims 100-108, wherein Z is selected from -NR X , -SX3, and -Ο- X3.
  111. 118. The compound or salt of any one of claims 100-117, wherein Z is selected from -OH, -SH, O-Ci-io alkyl, -NH(Cmo alkyl), and -NH2 β
  112. 119. The compound or salt of any one of claims 100-108, wherein -X is a represented by the
    O
    Λ peptide β
  113. 120. The compound or salt of claim 100-119, wherein -X is represented by the formula:
    Au
    N peptide—RX
    H , wherein RX comprises a reactive moiety.
  114. 121. The compound or salt of claim 121, wherein the reactive moiety comprises a maleimide.
  115. 122. The compound or salt of claim 100-119, wherein -X3 is represented by the formula:
    o χ
    N peptide-RX*- Antibody
    H ', wherein RX is a reactive moiety that has reacted with a moiety on an antibody to form an antibody construct immune-stimulatory compound conjugate.
    -281-
  116. 123. The compound or salt of claim 100-119, wherein -X3 is represented by the formula:
    O O 1-4 H , wherein RX is a reactive moiety.
  117. 124. The compound or salt of claim 124, wherein the reactive moiety is a maleimide.
    β
  118. 125. The compound or salt of any one of claims 100-119, wherein -X is represented by the
    O O formula: 1-4 H , wherein RX is a reactive moiety that has reacted with a moiety on an antibody to form an antibody construct immune-stimulatory conjugate.
  119. 126. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o o
    or a pharmaceutically acceptable salt thereof.
  120. 127. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
  121. 128. The compound or salt of claimlOO, wherein the compound is represented by the formula:
    acceptable salt thereof.
    o or a pharmaceutically
    -282129. The compound or salt of claim 100, wherein the compound is represented by the formula:
    X1O AA,
    ...I Il /> u ο o
    H Qh3 ο or a pharmaceutically acceptable salt o
    thereof.
  122. 130. The compound or salt of claim 100, wherein the compound is represented by the formula:
    CHs , or a pharmaceutically acceptable salt thereof.
  123. 131. The compound or salt of claim 100, wherein the compound is represented by the formula:
    H CH3 O , or a pharmaceutically acceptable salt thereof.
  124. 132. The compound or salt of claim 100, wherein the compound is represented by the formula:
    <> 11 I γΐθ μ-Ά Χ·Λ n 7 ν ΝΗ2
    Μ Ά1 . J. II /) TV HV% ο-^ο^ΝΓο
    F F
    F or a pharmaceutically acceptable salt thereof.
    -283133. The compound or salt of claim 100, wherein the compound is represented by the formula:
    0 , or a pharmaceutically acceptable salt thereof.
  125. 134. The compound or salt of claim 100, wherein the compound is represented by the formula:
    0 , or a pharmaceutically acceptable salt thereof.
  126. 135. The compound or salt of claim 100, wherein the compound is represented by the formula: o θ , or a pharmaceutically acceptable salt thereof.
  127. 136. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
    -284137. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
  128. 138. The compound or salt of claim 100, wherein the compound is represented by the formula: o o , or a pharmaceutically acceptable salt thereof.
  129. 139. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
  130. 140. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
    -285141. The compound or salt of claim 100, wherein the compound is represented by the formula:
    θ , or a pharmaceutically acceptable salt thereof.
  131. 142. The compound or salt of claim 100, wherein the compound is represented by the formula:
    ° , or a pharmaceutically acceptable salt thereof.
  132. 143. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
  133. 144. The compound or salt of claim 100, wherein the compound is represented by the formula:
    ° , or a pharmaceutically acceptable salt thereof.
    -286145. The compound or salt of claim 100, wherein the compound is represented by the formula:
    actable salt thereof.
  134. 146. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
  135. 147. The compound or salt of claim 100, wherein the compound is represented by the formula:
    ° , or a pharmaceutically acceptable salt thereof.
  136. 148. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
    -287149. The compound or salt of claim 100, wherein the compound is represented by the formula:
    ° , or a pharmaceutically acceptable salt thereof.
  137. 150. The compound or salt of claim 100, wherein the compound is represented by the formula:
    θ , or a pharmaceutically acceptable salt thereof.
  138. 151. The compound or salt of claim 100, wherein the compound is represented by the formula:
    Hi
    H o , or a pharmaceutically acceptable salt thereof.
  139. 152. The compound or salt of claim 100, wherein the compound is represented by the formula:
    o , or a pharmaceutically acceptable salt thereof.
    -288289
    Intellectual
    Property
    Office
    Application No: GB1620828.2
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