WO2022206753A1 - ANTICORPS GARP/TGFβ1 ET SON UTILISATION - Google Patents

ANTICORPS GARP/TGFβ1 ET SON UTILISATION Download PDF

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WO2022206753A1
WO2022206753A1 PCT/CN2022/083667 CN2022083667W WO2022206753A1 WO 2022206753 A1 WO2022206753 A1 WO 2022206753A1 CN 2022083667 W CN2022083667 W CN 2022083667W WO 2022206753 A1 WO2022206753 A1 WO 2022206753A1
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seq
antibody
sequence shown
antigen
binding
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PCT/CN2022/083667
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Chinese (zh)
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成广存
葛虎
曹卓晓
唐任宏
任晋生
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山东先声生物制药有限公司
江苏先声药业有限公司
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Priority to CN202280022651.3A priority Critical patent/CN117043187A/zh
Publication of WO2022206753A1 publication Critical patent/WO2022206753A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present disclosure relates to the field of antibodies, in particular, to GARP/TGF ⁇ 1 antibodies and applications thereof.
  • TGF ⁇ 1 Transforming growth factor ⁇ 1
  • TGF ⁇ 1 is a member of the TGF ⁇ protein family.
  • TGF ⁇ contains three specific isoforms, TGF ⁇ 1, TGF ⁇ 2, and TGF ⁇ 3, each of which is encoded by a separate gene.
  • TGF ⁇ is first produced as a precursor (pro-TGF-b) and converted into a latent protein by furin proprotein convertase in cells.
  • the latent protein TGF ⁇ is composed of N-terminal LAP protein and C-terminal mature TGF ⁇ in a non-covalent manner.
  • Latent TGF ⁇ cannot bind to receptors and activate downstream signaling pathways.
  • the active form of TGF ⁇ exists in the form of a homodimer without LAP, with a molecular weight of about 25kd.
  • Mature TGF ⁇ factor induces receptor phosphorylation and activation of downstream p-SMAD2/3 signaling pathway by binding to receptors T ⁇ RI and T ⁇ RII.
  • TGF ⁇ The production of mature TGF ⁇ is tightly regulated in multiple steps.
  • LTBP1 and LTBP3 are mainly secreted and expressed by fibroblasts, and a large number of LTBP1/TGF ⁇ complexes and LTBP3/TGF ⁇ complexes exist in the extracellular matrix in a latent state. It is then activated by proteases such as MMP2, MMP9 and plasmin to become mature TGF ⁇ .
  • TGF ⁇ can also exist in membrane-bound form, and the membrane-bound TGF ⁇ complex includes two types: GARP/TGF ⁇ and LRRC33/TGF ⁇ complex.
  • GARP is mainly expressed in regulatory T cells (Treg) and is essential for regulating TGF ⁇ activation.
  • the GARP/TGF ⁇ complex was shown to be expressed in Treg cells, which then release mature TGF ⁇ under the action of integrins.
  • TGF ⁇ is a multifunctional cytokine that inhibits the proliferation of many cell types, including epithelial, endothelial, hematopoietic, and immune cells.
  • the data show that in a variety of human tumor types, the TGF ⁇ 1 subtype is dominant, and the activation of the TGF ⁇ signaling pathway is mainly mediated by TGF ⁇ 1. Excessive activation of TGF ⁇ and its signaling pathways will cause TGF ⁇ -related diseases, such as cancer, tumor, inflammation, fibrotic diseases and cardiovascular and cerebrovascular diseases.
  • TGF ⁇ 1 has important clinical value for the treatment of TGF ⁇ 1-related diseases.
  • the present disclosure provides antibody molecules that specifically bind to GARP/TGF ⁇ 1 complexes and/or monomers therein, thereby blocking the maturation process of TGF ⁇ 1 and reducing the release of active TGF ⁇ 1, which is of great significance for the treatment of TGF ⁇ 1-related diseases.
  • the present disclosure provides antibodies or antigen-binding fragments, multi-specific antigen-binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid molecules, vectors, cells, methods of manufacture, pharmaceutical compositions, pharmaceutical uses and diseases that bind to GARP/TGF ⁇ 1 complexes of treatment.
  • the present disclosure provides an antibody or antigen-binding fragment that binds a GARP/TGF ⁇ 1 complex, the antibody or antigen-binding fragment comprising HCDR1-3, and/or LCDR1-3, the HCDR1-3 and/or The LCDR1-3 respectively comprise sequences shown in Table 2 or Table 15, or sequences with at least 70% identity or at most 5 mutations with the sequences shown in Table 2 or Table 15;
  • the HCDR1 comprises SEQ ID NO: 104, 107, 109, 117, 120, 122, 130, 133, 135, 143, 146, 148, 156, 159, 161, 169, 172 or 174. sequence, or a sequence with at least 70% identity or at most 5 mutations therewith;
  • the HCDR2 comprises SEQ ID NOs: 105, 182-186, 108, 110, 118, 121, 123, 131, 134, 136, 144, 147, 149, 157, 188-189, 160, 162, 170 , 173 or 175, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the HCDR3 comprises, or is at least 70% identical to, the sequence set forth in any one of SEQ ID NOs: 106, 111, 119, 124, 132, 137, 145, 150, 158, 163, 171 or 176 or Up to 5 mutated sequences;
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 112, 115, 125, 128, 138, 141, 151, 154, 164, 167, 177 or 180, or has at least 70% identity thereto or Up to 5 mutated sequences;
  • the LCDR2 comprises or has at least 70% of the sequence set forth in any one of SEQ ID NOs: 113, 116, 126, 129, 139, 142, 152, 187, 155, 165, 168, 178 or 181 sequence of identity or up to 5 mutations;
  • the LCDR3 comprises the sequence set forth in any one of SEQ ID NOs: 114, 127, 140, 153, 166, 179, or a sequence having at least 70% identity or at most 5 mutations therewith.
  • the antibody or antigen-binding fragment comprises a sequence shown in any of groups (1)-(7):
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 104, 107 or 109;
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 105, 182-186, 108 or 110
  • the HCDR3 comprise the sequence shown in any one of SEQ ID NO: 106 or 111;
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 112 or 115
  • the LCDR2 comprises any one of SEQ ID NO: 113 or 116
  • the sequence shown in item, the LCDR3 comprises the sequence shown in SEQ ID NO: 114;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 117, 120 or 122
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 118, 121 or 123
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 119 or 124
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 125 or 128, and the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 126 or 129
  • the LCDR3 comprises the sequence shown in SEQ ID NO: 127;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 130, 133 or 135, the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 131, 134 or 136, and the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 132 or 137; and/or, the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 138 or 141, and the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 139 or 142 , the LCDR3 comprises the sequence shown in SEQ ID NO: 140;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 143, 146 or 148
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 144, 147 or 149
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 145 or 150
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 151 or 154
  • the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 152, 187 or 155
  • the sequence shown, the LCDR3 comprises the sequence shown in SEQ ID NO: 153;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 156, 159 or 161
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 157, 188-189, 160 or 162
  • the HCDR3 comprise the sequence shown in any one of SEQ ID NO: 158 or 163
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 164 or 167
  • the LCDR2 comprises any one of SEQ ID NO: 165 or 168
  • the sequence shown in item, the LCDR3 comprises the sequence shown in SEQ ID NO: 166;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 169, 172 or 174
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 170, 173 or 175
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 171 or 176
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 177 or 180
  • the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 178 or 181
  • the LCDR3 comprises the sequence shown in SEQ ID NO: 179;
  • the HCDR1-3 and/or the LCDR1-3 are determined according to the rules of Kabat, Chothia or IMGT.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises the HCDRs 1-3, the light chain variable region zone contains the LCDR1-3;
  • the heavy chain variable region comprises: as SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 32-40, 41, 46-48, 49-51, 54-61, 68 , 70-72, 78, 81-87, 90, 93-99, or a sequence with at least 70% identity or at most 20 mutations to the sequence shown;
  • the light chain variable region comprises: as SEQ ID NO: 16, 18, 20, 22, 24, 26, 28-31, 42-45, 52-53, 62-67, 69, 73-77 , 79-80, 88-89, 91-92, 100-103 any one of the sequences shown, or a sequence that is at least 70% identical to the sequence shown or has at most 20 mutations;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 15, 27, 32-40, and the light chain variable region comprises the sequence shown in SEQ ID NO: 16, 28 The sequence shown in any one of -31;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 17, 41, 46-48, and the light chain variable region comprises the sequence shown in SEQ ID NO: 18, 42 -45 any one of the sequences shown;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 19, 49-51, 54-61, and the light chain variable region comprises the sequence shown in SEQ ID NO: 20 , 52-53, the sequence shown in any one of 62-67;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 21, 68, 70-72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 22, 69 , the sequence shown in any one of 73-77;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 23, 78, 81-87;
  • the light chain variable region comprises the sequence shown in SEQ ID NO: 24, 79 The sequence shown in any one of -80, 88-89;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 25, 90, 93-99, and the light chain variable region comprises the sequence shown in SEQ ID NO: 26, 91 The sequence shown in any one of -92, 100-103;
  • the heavy chain variable region and/or the light chain variable region comprise at least 70% identity or at most 20 mutations to the sequences shown in groups (1)-(6). the sequence of.
  • the heavy chain variable region and/or the light chain variable region are selected from VH and/or VL shown in Tables 3, 5, 7, 9, 11 or 13; preferably, The heavy chain variable region and the light chain variable region are paired as in Tables 4, 6, 8, 10, 12 or 14.
  • the at least 70% identity is further preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99% or 100% identity; further preferably at most 5 mutations are at most 4, 3, 2, 1 or 0 mutations; further preferably at most 20 mutations are at most 19, 18, 17 , 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations; preferably, the mutations are insertions, deletions or substitutions, The substitutions are preferably conservative amino acid substitutions, and the mutations are preferably back mutations or hotspot mutations.
  • the antibody or antigen-binding fragment further comprises a heavy chain constant region and/or a light chain constant region;
  • the heavy chain constant region is selected from IgG, such as IgG1, IgG2, IgG3 or IgG4; the IgG can be selected from human IgG, such as human IgG1 or human IgG4; the light chain constant region is selected from kappa chain or lambda chain, preferably a kappa chain;
  • the heavy chain constant region comprises the sequence shown in SEQ ID NO: 9 or 10
  • the light chain constant region comprises the sequence shown in SEQ ID NO: 12 or 13.
  • the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies Antibodies, Multivalent Antibodies, Whole Antibodies, Fragments of Whole Antibodies, Naked Antibodies, Conjugated Antibodies, Chimeric Antibodies, Humanized Antibodies, Fully Human Antibodies, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody or single domain antibody.
  • the antibody or antigen-binding fragment further includes a conjugate;
  • the conjugate can be selected from a therapeutic agent or a tracer, and the therapeutic agent can be selected from a radioisotope, a chemotherapeutic agent, or an immune Modulators,
  • the tracers may be selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photosensitizers.
  • the antibody or antigen-binding fragment binds the human GARP/TGF ⁇ 1 complex and/or the monkey GARP/TGF ⁇ 1 complex;
  • the KD value of the antibody or antigen-binding fragment for binding to human GARP/TGF ⁇ 1 complex is less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M;
  • the KD value of the antibody or antigen-binding fragment for binding to the monkey GARP/TGF ⁇ 1 complex is less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M.
  • the antibody or antigen-binding fragment further has a binding characteristic selected from one of the group consisting of (1) binding to (human) GARP monomer and not binding to (human) TGF ⁇ 1 monomer; (2) Binds (human) TGF ⁇ 1 monomer, does not bind (human) GARP monomer, preferably, its KD value for binding to (human) TGF ⁇ 1 monomer is less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E -11M, 1E-12M or 1E-13M; (3) only binds (human) GARP/TGF ⁇ 1 complex, not (human) GARP monomer and (human) TGF ⁇ 1 monomer;
  • the binding site of the antibody or antigen-binding fragment has one of the characteristics selected from the group consisting of: (1) the binding site is located in a (human) GARP monomer; (2) the binding site is located in a (human) TGF ⁇ 1 monomer (3) The binding site is located on the (human) GARP/TGF ⁇ 1 complex, not only on GARP or TGF ⁇ 1 of the complex.
  • the antibody or antigen-binding fragment further has at least one characteristic selected from the group consisting of: (1) binding to (human) GARP/TGF ⁇ 1 complex protein; (2) binding to express (human) GARP /TGF ⁇ 1 complex cells, such as (human) Treg cells; (3) blocking the formation of activated TGF ⁇ 1; (4) inhibiting (human) Treg cell function, such as inhibiting SMAD2 protein phosphorylation.
  • the present disclosure provides an antibody or antigen-binding fragment that binds to the GARP/TGF ⁇ 1 complex, GARP or TGF ⁇ 1, the antibody or antigen-binding fragment comprising HCDR1-3, and/or LCDR1-3, the HCDR1- 3 and/or the LCDR1-3 respectively comprise sequences shown in Table 2 or Table 15, or sequences with at least 70% identity or at most 5 mutations with the sequences shown in Table 2 or Table 15; preferably, the HCDR1 -3 and/or said LCDR1-3 are determined according to Kabat, Chothia or IMGT rules.
  • the antibody or antigen-binding fragment binds the GARP/TGF ⁇ 1 complex
  • the HCDR1 comprises a sequence set forth in one of SEQ ID NO: 130, 133, 135, 143, 146, 148, 169, 172 or 174, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the HCDR2 comprises, or is at least 70% identical to, the sequence set forth in any one of SEQ ID NOs: 131, 134, 136, 144, 147, 149, 157, 187-188, 160, 162, 170, 173 or 175 or up to 5 mutated sequences;
  • the HCDR3 comprises a sequence set forth in any one of EQ ID NOs: 132, 137, 145, 150, 158, 163, 171 or 176, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the LCDR1 comprises a sequence set forth in any one of SEQ ID NOs: 138, 141, 151, 154, 164, 167, 177 or 180, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the LCDR2 comprises a sequence set forth in any one of SEQ ID NOs: 139, 142, 152, 187, 155, 165, 168, 178 or 181, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the LCDR3 comprises the sequence shown in any one of SEQ ID NOs: 140, 153, 179, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the antibody or antigen-binding fragment comprises the sequences shown in any of groups (1)-(4):
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 130, 133 or 135, the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 131, 134 or 136, and the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 132 or 137; and/or, the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 138 or 141, and the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 139 or 142 , the LCDR3 comprises the sequence shown in SEQ ID NO: 140;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 143, 146 or 148
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 144, 147 or 149
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 145 or 150
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 151 or 154
  • the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 152, 187 or 155
  • the sequence shown, the LCDR3 comprises the sequence shown in SEQ ID NO: 153;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 169, 172 or 174
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 170, 173 or 175
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 171 or 176
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 177 or 180
  • the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 178 or 181
  • the LCDR3 comprises the sequence shown in SEQ ID NO: 179;
  • the antibody or antigen-binding fragment specifically binds GARP
  • the HCDR1 comprises a sequence set forth in one of SEQ ID NOs: 104, 107, 109, 156, 159 or 161, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the HCDR2 comprises the sequence set forth in any one of SEQ ID NOs: 105, 182-186, 108, 110, 157, 188-189, 160 or 162, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the HCDR3 comprises a sequence set forth in any one of EQ ID NO: 106, 111, 158 or 163, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the LCDR1 comprises the sequence set forth in any one of SEQ ID NOs: 112, 115, 164 or 167, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the LCDR2 comprises a sequence set forth in any one of SEQ ID NO: 113, 116, 165 or 168, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the LCDR3 comprises the sequence set forth in any one of SEQ ID NO: 114 or 166, or a sequence having at least 70% identity or at most 5 mutations therewith;
  • the antibody or antigen-binding fragment comprises a sequence shown in any of groups (1)-(3):
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 104, 107 or 109;
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 105, 182-186, 108 or 110
  • the HCDR3 comprise the sequence shown in any one of SEQ ID NO: 106 or 111;
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 112 or 115
  • the LCDR2 comprises any one of SEQ ID NO: 113 or 116
  • the sequence shown in item, the LCDR3 comprises the sequence shown in SEQ ID NO: 114;
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 156, 159 or 161
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 157, 188-189, 160 or 162
  • the HCDR3 comprise the sequence shown in any one of SEQ ID NO: 158 or 163
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 164 or 167
  • the LCDR2 comprises any one of SEQ ID NO: 165 or 168
  • the sequence shown in item, the LCDR3 comprises the sequence shown in SEQ ID NO: 166;
  • the antibody or antigen-binding fragment specifically binds TGF ⁇ 1 and comprises a sequence shown in any of groups (1)-(2):
  • the HCDR1 comprises the sequence shown in any one of SEQ ID NO: 117, 120 or 122
  • the HCDR2 comprises the sequence shown in any one of SEQ ID NO: 118, 121 or 123
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NO: : the sequence shown in any one of 119 or 124
  • the LCDR1 comprises the sequence shown in any one of SEQ ID NO: 125 or 128, and the LCDR2 comprises the sequence shown in any one of SEQ ID NO: 126 or 129
  • the LCDR3 comprises the sequence shown in SEQ ID NO: 127;
  • the antibody or antigen-binding fragment comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises the HCDRs 1-3, the light chain variable region zone contains the LCDR1-3;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 15, 27, 32-40, and the light chain variable region comprises the sequence shown in SEQ ID NO: 16, 28- The sequence shown in any one of 31;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 17, 41, 46-48, and the light chain variable region comprises the sequence shown in SEQ ID NO: 18, 42- 45 any one of the sequences shown;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NO: 19, 49-51, 54-61, and the light chain variable region comprises the sequence shown in SEQ ID NO: 20, The sequence shown in any one of 52-53 and 62-67;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 21, 68, 70-72, and the light chain variable region comprises the sequence shown in SEQ ID NO: 22, 69, The sequence shown in any one of 73-77;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 23, 78, 81-87;
  • the light chain variable region comprises the sequence shown in SEQ ID NO: 24, 79- The sequence shown in any one of 80 and 88-89;
  • the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 25, 90, 93-99, and the light chain variable region comprises the sequence shown in SEQ ID NO: 26, 91- 92, the sequence shown in any one of 100-103;
  • the heavy chain variable region and/or the light chain variable region comprises at least 70% identity or at most 20 mutations to the sequences shown in groups (1)-(6). sequence.
  • the heavy chain variable region and/or the parent chain variable region are selected from the VH and/or VL shown in Tables 3, 5, 7, 9, 11 or 13; preferably, the The heavy chain variable region and the light chain variable region are paired as in Tables 4, 6, 8, 10, 12 or 14.
  • the at least 70% identity is further preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99% or 100% identity; further preferably at most 5 mutations are at most 4, 3, 2, 1 or 0 mutations; further preferably at most 20 mutations are at most 19, 18, 17 , 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations; preferably, the mutations are insertions, deletions or substitutions, The substitutions are preferably conservative amino acid substitutions, and the mutations are preferably back mutations or hotspot mutations.
  • the antibody or antigen-binding fragment further comprises a heavy chain constant region and/or a light chain constant region;
  • the heavy chain constant region is selected from IgG, such as IgGl, IgG2, IgG3 or IgG4, which may be selected from human IgG, such as human IgGl or human IgG4, and the light chain constant region is selected from kappa chain or lambda chain, preferably a kappa chain;
  • the heavy chain constant region comprises the sequence shown in SEQ ID NO:9
  • the light chain constant region comprises the sequence shown in SEQ ID NO:12.
  • the antibody or antigen-binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies Antibodies, Multivalent Antibodies, Whole Antibodies, Fragments of Whole Antibodies, Naked Antibodies, Conjugated Antibodies, Chimeric Antibodies, Humanized Antibodies, Fully Human Antibodies, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabody or single domain antibody.
  • the antibody or antigen-binding fragment further includes a conjugate;
  • the conjugate can be selected from a therapeutic agent or a tracer, and the therapeutic agent can be selected from a radioisotope, a chemotherapeutic agent, or an immune Modulators,
  • the tracers may be selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, and photosensitizers.
  • the antibody or antigen-binding fragment further has at least one characteristic selected from the group consisting of: (1) binding to (human) GARP/TGF ⁇ 1 complex protein; (2) binding to express (human) GARP /TGF ⁇ 1 complex cells, such as (human) Treg cells; (3) block the formation of activated TGF ⁇ 1; (4) inhibit (human) Treg cell function, for example, inhibit SMAD2 protein phosphorylation.
  • the present disclosure provides a multispecific antigen-binding molecule comprising at least a first antigen-binding moiety and a second antigen-binding moiety, the first antigen-binding moiety comprising the aforementioned antibody or An antigen-binding fragment, the second antigen-binding moiety binds to other targets different from the first antigen-binding moiety or to a different epitope of the same target; preferably, the second antigen-binding moiety is an antibody or an antigen-binding fragment;
  • the other targets are selected from the group consisting of: (1) tumor-specific antigens (TSA) or tumor-associated antigens (TAA), such as CD24; (2) immune checkpoints, such as PD-1 or PD-L1; (3) Targets that recruit and/or activate immune cells, such as CD3 or CD16.
  • TSA tumor-specific antigens
  • TAA tumor-associated antigens
  • CD24 tumor-associated antigens
  • immune checkpoints such as PD-1 or PD-L1
  • Targets that recruit and/or activate immune cells such as CD3 or CD16.
  • the present disclosure provides a chimeric antigen receptor (CAR) comprising at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the cell External antigen-binding domains include the aforementioned antibodies or antigen-binding fragments or the aforementioned multispecific antigen-binding molecules.
  • CAR chimeric antigen receptor
  • the present disclosure provides an immune effector cell expressing the aforementioned chimeric antigen receptor and/or comprising a nucleic acid molecule encoding the aforementioned chimeric antigen receptor;
  • the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells, more preferably, The T cells are selected from cytotoxic T cells, regulatory T cells or helper T cells;
  • the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
  • the present disclosure also provides an isolated nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, or chimeric antigen receptor.
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid molecule.
  • the present disclosure also provides a cell comprising the aforementioned vector.
  • the present disclosure also provides a method for preparing the aforementioned antibody or antigen-binding fragment, or a multispecific antigen-binding molecule, the method comprising: (1) culturing the aforementioned cells and/or (2) isolating the cells Expressed antibodies or antigen-binding fragments, or multispecific antigen-binding molecules.
  • the present disclosure also provides a method for preparing the aforementioned immune effector cells, the method comprising introducing a nucleic acid molecule encoding the aforementioned chimeric antigen receptor into the immune effector cells, and/or activating the immune effector cells The chimeric antigen receptor is expressed.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding molecule, multispecific antigen-binding molecule, immune effector cell, nucleic acid molecule, vector, cell or prepared according to the aforementioned method
  • the resulting product preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.
  • the present disclosure also provides the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, immune effector cells, nucleic acid molecules, vectors, cells, pharmaceutical compositions, or products prepared according to the aforementioned methods in preparation for treatment Use in medicine for TGF ⁇ -related diseases; preferably, the TGF ⁇ -related diseases are selected from cancer, fibrotic diseases, inflammation, cardiovascular and cerebrovascular diseases or chronic infectious diseases.
  • the present disclosure also provides a method of treating a TGF ⁇ -related disease, the method comprising administering to a subject an effective amount of a drug comprising the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule , immune effector cells, nucleic acid molecules, vectors, cells, pharmaceutical compositions or products prepared according to the aforementioned methods, preferably, the TGF ⁇ -related diseases are selected from cancer, fibrotic diseases, inflammation, cardiovascular and cerebrovascular diseases or chronic infectious diseases disease.
  • the present disclosure also provides the aforementioned antibody or antigen-binding fragment multispecific antigen-binding molecule, immune effector cell, nucleic acid molecule, vector, cell, pharmaceutical composition or product prepared according to the aforementioned method, for use in therapy
  • the medicine for TGF ⁇ -related diseases preferably, the TGF ⁇ -related diseases are selected from cancer, fibrotic diseases, inflammation, cardiovascular and cerebrovascular diseases or chronic infectious diseases.
  • compositions comprising A and B
  • composition containing other components in addition to A and B all fall within the scope of the aforementioned "a composition”.
  • TGF ⁇ 1 refers to transforming growth factor ⁇ 1, which is a member of the TGF ⁇ 1 family, including pro-form (pro-TGF ⁇ 1), latent form (latent TGF ⁇ 1), and mature form (mature TGF ⁇ 1).
  • TGF ⁇ 1 described in the present disclosure can be derived from mammals, eg, humans, non-human primates, rodents, eg, human TGF ⁇ 1, cynomolgus TGF ⁇ 1, and murine TGF ⁇ 1, the sequences of which include, but do not Limited to UniProt number: P01137, Uniprot: A0A2K5TJB2, Uniprot: P04202.
  • GARP glycoprotein A repetitions predominant
  • the GARPs described in the present disclosure may be derived from mammals, eg, humans, non-human primates, rodents, eg, human GARP, cynomolgus GARP, and murine GARP, the protein sequences of which include, but are not limited to, UniProt No.: Q14392, Uniprot No.: A0A2K5X2X0, Uniprot: G3XA59.
  • the terms "GARP/TGF ⁇ 1 complex", “GARP complex” or “GARP complex” in the present disclosure all refer to a complex consisting of GARP and proTGF ⁇ 1, or a complex consisting of GARP and latent TGF ⁇ 1.
  • LRRC33 (Leucine-Rich Repeat-Containing Protein 33) of the present disclosure refers to leucine-rich repeat protein 33, which belongs to the TGF ⁇ 1 receptor and is capable of forming the LRRC33/TGF ⁇ 1 complex therewith.
  • the LRRC33s described in the present disclosure may be derived from mammals, eg, humans, non-human primates, rodents, eg, human LRRC33, cynomolgus LRRC33, or murine LRRC33, the protein sequences of which include, but are not limited to, UniProt No.: Q86YC3.
  • LRRC33/TGF ⁇ 1 complex refers to a complex consisting of LRRC33 and proTGF ⁇ 1 or a complex consisting of LRRC33 and latent TGF ⁇ 1.
  • LTBP1 Latent Transforming Growth Factor Beta Binding Protein 1
  • latent transforming growth factor beta binding protein 1 belongs to the TGF ⁇ 1 receptor and can form a LTBP1/TGF ⁇ 1 complex with it.
  • the LTBP1 described in the present disclosure can be derived from mammals, eg, humans, non-human primates, rodents, eg, human LTBP1, cynomolgus LTBP1, and murine LTBP1, the protein sequences of which include, but are not limited to, UniProt Number: Q14766.
  • LTBP1/TGF ⁇ 1 complex refers to a complex consisting of LTBP1 and pro TGF ⁇ 1 or a complex consisting of LTBP1 and latent TGF ⁇ 1.
  • the term "specifically binds" in the present disclosure refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
  • high affinity generally refers to having about 10-7 M or less, about 10-8 M or less, about 1 ⁇ 10-9 M or less, about 1 ⁇ 10-10 M or less, KD of 1 ⁇ 10-11 M or lower or 1 ⁇ 10-12 M or lower.
  • Equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis method, for example, see the method for obtaining KD value shown in Example 8 of the present disclosure.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody.
  • antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.
  • antibody refers to an antibody comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen.
  • the “antibodies” of the present disclosure encompass various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • “Antibody” of the present disclosure includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody of the present disclosure includes a typical "quad-chain antibody”, which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that In the N-terminal to C-terminal direction consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and , when the full-length antibody is of the IgE isotype, optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain heavy chain constant region
  • CH3 domain heavy chain constant region
  • the full-length antibody is of the IgE isotype
  • immunoglobulins A polypeptide chain composed of a constant region (CL); the heavy chain and the heavy chain and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure. Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" of the present disclosure can be divided into five classes, or isotypes referred to as immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , respectively. chain, gamma chain, alpha chain and epsilon chain.
  • Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Antibody of the present disclosure also includes antibodies that do not contain a light chain, such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca (Vicugnapacos) and other heavy chain antibodies (heavy-chain antibodies, HCAbs) and in sharks and other cartilaginous fish found in the new immunoglobulin receptors (Ig new antigen receptor, IgNAR).
  • a light chain such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca (Vicugnapacos) and other heavy chain antibodies (heavy-chain antibodies, HCAbs) and in sharks and other cartilaginous fish found in the new immunoglobulin receptors (Ig new antigen receptor, IgNAR).
  • the "antibodies” of the present disclosure may be derived from any animal, including, but not limited to, humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelid, ram Camelids, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • humans and non-human animals which may be selected from primates, mammals, rodents, and vertebrates, such as camelid, ram Camelids, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • Antibody of the present disclosure includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked Antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.
  • the term "monoclonal antibody” of the present disclosure refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for possible variants (eg, containing a naturally occurring mutation or generated during the manufacture of a formulation, such variants typically except that they are present in small amounts), the individual antibodies comprising the population are identical and/or bind the same epitope.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method.
  • monoclonal antibodies can be made by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology, and the use of transgenic animals that contain all or part of the human immunoglobulin loci method and other methods known in the art.
  • natural antibody refers to an antibody that is produced and paired by the immune system of a multicellular organism.
  • engineered antibody refers to a non-natural antibody obtained by techniques such as genetic engineering, antibody engineering, etc. Bispecific antibodies and more.
  • the term "monospecific antibody” of the present disclosure refers to an antibody having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific antibody of the present disclosure refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or different from a different antigen. Epitope binding.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valency of the present disclosure refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
  • monovalent antibody bivalent antibody
  • tetravalent antibody hexavalent antibody
  • hexavalent antibody refer to one binding site, two binding sites, four binding sites and Antibodies with six binding sites present.
  • full-length antibody “intact antibody,” and “intact antibody” are used interchangeably in this disclosure and refer to a structure that is substantially similar to that of a native antibody.
  • antigen-binding fragment and “antibody fragment” are used interchangeably in this disclosure, which do not possess the full structure of an intact antibody, but only include partial or partial variants of the intact antibody, which partial or partial variants Possess the ability to bind antigens.
  • Antigen-binding fragments or “antibody fragments” of the present disclosure include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, diabodies, and single domain antibodies.
  • Papain digestion of intact antibodies produces two identical antigen-binding fragments, termed "Fab” fragments, each containing the heavy and light chain variable domains, as well as the light chain constant domain and the heavy chain first constant domain (CH1 ).
  • Fab fragment refers to a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by adding a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domains carry free thiol groups. Pepsin treatment produces an F(ab') 2 fragment with two antigen binding sites (two Fab fragments) and a portion of the Fc region.
  • Fd refers to an antibody consisting of VH and CH1 domains.
  • Fv refers to antibody fragments consisting of one-armed VL and VH domains. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (eg, an Fd fragment, which contains only three CDRs specific for the antigen) is able to recognize and bind the antigen, albeit perhaps with lower affinity than the intact binding site.
  • scFv single-chain variable fragment of the present disclosure refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Roseburg and Moore, Springer-Verlag , New York, pp. 269-315 (1994)).
  • Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • GGGGS linker with the amino acid sequence
  • Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res.
  • a disulfide bond may also exist between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).
  • diabody of the present disclosure, whose VH and VL domains are expressed on a single polypeptide chain, but uses linkers that are too short to allow pairing between the two domains of the same chain, forcing the domains Pairs with the complementary domains of the other chain and creates two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al. , Structure 2: 1121-1123 (1994)).
  • single domain antibody (sdAb), “VHH” and “nanobody” of the present disclosure have the same meaning and are used interchangeably and refer to the variable region of a cloned antibody heavy chain, constructed solely by A single-domain antibody consisting of a heavy chain variable region that is the smallest fully functional antigen-binding fragment.
  • an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
  • Single domain antibodies can be derived from camelid heavy chain antibodies or from cartilaginous IgNARs.
  • naked antibody of the present disclosure refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term “conjugated antibody” refers to an antibody that is conjugated to a therapeutic agent or tracer.
  • chimeric antibody refers to an antibody in which a portion of its light or/and heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), but in any case it remains Binding activity to target antigens (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody can include antibodies (eg, human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a primary antibody (eg, a murine antibody) and the heavy and The light chain constant region is derived from a second antibody (eg, a human antibody).
  • a primary antibody eg, a murine antibody
  • a second antibody eg, a human antibody
  • humanized antibody refers to a genetically engineered, non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.
  • Fully human antibody of the present disclosure refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” of the present disclosure do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region in the present disclosure refers to the region of the heavy or light chain of an antibody involved in binding an antibody to an antigen. Region” is used interchangeably with “VL", “LCVR”.
  • VH and VL respectively
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably in this disclosure and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is due to the spatial structure of the region. It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • the light chain variable region CDR can be abbreviated as LCDR.
  • framework region or "FR region” are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody.
  • the "CDRs" of the present disclosure may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare .cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign .cgi#results).
  • the CDRs of the present disclosure include overlaps and subsets of amino acid residues differently defined.
  • Kabat numbering system generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system generally refers to the numbering system based on The International ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol .27:55-77, 2003.
  • IMGT International ImMunoGeneTics information system
  • heavy chain constant region refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the antibody's Variable domains have more conserved amino acid sequences.
  • a “heavy chain constant region” comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from a CH1, Fc or CH3 domain.
  • light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the papain hydrolyzed carboxy-terminal portion of an antibody of an intact antibody, which typically comprises the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, thus, the Fc region can include or excluding Lys447.
  • amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids within each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions:
  • identity of the present disclosure can be calculated by aligning the sequences for optimal comparison purposes (e.g., which may be the most optimal) in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences. gaps may be introduced in either or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences varies with the identical positions shared by the sequences.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com), which has been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or The PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com) using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between the two nucleotide sequences.
  • a particularly preferred set of parameters is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described in the present disclosure can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • search can be performed, for example, using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST the default parameters of the corresponding programs. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor of the present disclosure refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen-binding domain, such as The variable heavy or light chain of the antibody, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • nucleic acid of the present disclosure includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is generally represented as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and A mixed polymer of two or more of these molecules.
  • DNA deoxyribonucleic acid
  • cDNA complementary DNA
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described in the present disclosure may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present disclosure in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg, cDNA
  • RNA eg, mRNA
  • the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
  • An "isolated" nucleic acid of the present disclosure refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to in this disclosure as "expression vectors.”
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included in the present disclosure are mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell.
  • composition refers to a formulation that is in a form that allows for the biological activity of the active ingredients contained therein to be effective and that does not contain substances that would be unacceptable to the subject to whom the pharmaceutical composition is administered. Toxic additional ingredients.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow (reduce) undesired physiological changes or lesions, such as the progression of cancer, in the subject being treated.
  • Beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
  • alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
  • subject of the present disclosure refers to an organism receiving treatment for a particular disease or disorder as described in the present disclosure.
  • subjects and patients include mammals, such as humans, primates (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • an effective amount of the present disclosure refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease.
  • Effective amount also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” of the present disclosure refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to in this disclosure.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a half-way response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • Figure 1 Flow cytometry analysis to detect the expression of GARP in hGARP-293T cells, the stably transfected cell line, the ordinate is the number of cells, and the abscissa is the fluorescence intensity;
  • Figure 2 Flow cytometry analysis to detect the expression of GARP in hGARP complex-293T cells, the stably transfected cell line, the ordinate is the number of cells, and the abscissa is the fluorescence intensity;
  • Figure 3 Flow cytometry analysis to detect GARP expression in hGARP complex-CHOK1/S cells stably transfected cell line, the ordinate is the number of cells, and the abscissa is the fluorescence intensity;
  • Figure 4 Flow cytometry analysis to detect the expression of integrin ⁇ V ⁇ 6-293T cells, the ordinate is the number of cells, and the abscissa is the fluorescence intensity;
  • ELISA detects the serum titer of mice after immunization, where the ordinate is OD450, and the abscissa is the serum dilution;
  • Figure 5A is the titer of the SJL mouse 6 immune serum, and
  • Figure 5B is the Babl/c mouse 6 immune serum titer price;
  • ELISA detects the binding of murine antibody Mab017 to human GARP, human GARP/TGF ⁇ 1 complex, human proTGF ⁇ 1 and human LTBP1/TGF ⁇ 1 complex, respectively;
  • ELISA detects the binding of murine antibody Mab087 to human GARP, human GARP/TGF ⁇ 1 complex, human proTGF ⁇ 1 and human LTBP1/TGF ⁇ 1 complex, respectively;
  • ELISA detects the binding of mouse antibody Mab138 to human GARP, human GARP/TGF ⁇ 1 complex, human proTGF ⁇ 1 and human LTBP1/TGF ⁇ 1 complex, respectively;
  • FIGS. 9A to 9D ELISA detects the binding of mouse antibody Rab171 to human GARP, human GARP/TGF ⁇ 1 complex, human proTGF ⁇ 1 and human LTBP1/TGF ⁇ 1 complex, respectively;
  • ELISA detects the binding of chimeric antibody ch-Mab195 to human GARP and human GARP/TGF ⁇ 1 complexes
  • ELISA detects the binding of chimeric antibody ch-Mab201 to human GARP/TGF ⁇ 1 complex
  • ELISA detects the binding ability of humanized antibody to human GARP-his protein
  • ELISA detects the binding ability of humanized antibody to human GARP/TGF ⁇ 1 complex
  • ELISA detects the binding ability of humanized antibody to human LTBP1/TGF ⁇ 1 complex
  • ELISA detects the binding ability of humanized antibody h087-H2L2 to human LRRC33/TGF ⁇ 1 complex
  • ELISA detects the binding ability of humanized antibody to cynomolgus monkey GARP/TGF ⁇ 1 complex
  • FACS detects the binding of humanized antibody to cynomolgus monkey GARP/TGF ⁇ 1 complex at the cellular level
  • FIGS. 19A-19B FACS detects the binding of humanized antibodies to human GARP-293T cells or human GARP-complex-293T cells;
  • FIGS. 21A-21B flow cytometry analysis detects the binding of humanized antibodies to human Treg cells
  • luciferase reporter system detects the inhibitory effect of antibody on TGF- ⁇ 1 secretion
  • Figures 23A-23D the percentage of Treg cells pSMAD2
  • Fig. 24A-Fig. 24B anti-hGARP/TGF ⁇ 1 antibody inhibits human Treg function in vivo, wherein Fig. 24A is the result of GVHD score, and Fig. 24B is the result of survival rate;
  • Figure 26 Human GARP transgenic mice pharmacokinetics.
  • h017-H2dL2, GARP-h017-H2dL2 and GARP-hu017-H2dL2 shown in the following examples and accompanying drawings all represent the same humanized antibody
  • h087-H2L2, GARP-h087-H2L2 and GARP-hu087 -H2L2 all represent the same humanized antibody
  • h138-H8L5, GARP-h138-H8L5, GARP-hu138-H8L5 all represent the same humanized antibody
  • h171-H3L5, GARP-h171-H3L5 and GARP-hu171-H3L5 all represent the same humanized antibody
  • h195-H3L1, GARP-h195-H3L1, GARP-hu195-H3L1 all represent the same humanized antibody
  • h201-H5L3, GARP-h201-H5L3, GARP-hu201-H5L3 all represent the same humanized
  • the GARP and proTGF ⁇ 1 plasmids were separately transfected into HEK293 cells to obtain the corresponding monomeric proteins; GARP and proTGF ⁇ 1 plasmids were mixed and co-transfected to obtain the GARP/TGF ⁇ 1 complex.
  • the preparation method of cynomolgus monkey and mouse protein is the same as the preparation method of human recombinant protein.
  • the cynomolgus monkey GARP sequence is from Uniprot number: A0A2K5X2X0, and the TGF ⁇ 1 precursor protein sequence is from Uniprot: A0A2K5TJB2.
  • the mouse GARP sequence was from Uniprot: G3XA59, and the TGF ⁇ 1 precursor protein sequence was from Uniprot: P04202.
  • the specific sequence information of the GARP and proTGF ⁇ 1 recombinant proteins is shown below:
  • Human GARP-his protein (his-tagged human GARP protein extracellular domain fusion protein) (SEQ ID NO: 1):
  • Human proTGF ⁇ 1-his protein (his-tagged human TGF ⁇ 1 precursor fusion protein) (SEQ ID NO: 2):
  • Cynomolgus GARP-Flag/his (Flag/his-tagged cynomolgus GARP extracellular domain fusion protein) (SEQ ID NO: 3):
  • Cynomolgus monkey proTGF ⁇ 1 (untagged cynomolgus monkey TGF ⁇ 1 precursor protein sequence) (SEQ ID NO: 4):
  • His-mTGF ⁇ 1-pro His-tagged mouse TGF ⁇ 1 precursor protein (SEQ ID NO: 6):
  • Recombinant proteins constructed according to the amino acid sequences of human LRRC33 and LTBP1 using human LRRC33 protein (UniProt number: Q86YC3) and human LTBP1 protein (UniProt number: Q14766) as template sequences, a fusion protein with his tag was designed and cloned into the pTT5 vector, respectively. Then the plasmids of LRRC33 and LTBP1 and the aforementioned proTGF ⁇ 1 plasmid were co-transfected into HEK293 cells. After seven days of culture, the cell supernatant was collected and purified to obtain human LRRC33/TGF ⁇ 1 complex and human LTBP1/TGF ⁇ 1 complex (also known as human LTBP1 complex).
  • the specific sequence information of the recombinant protein is as follows:
  • hLRRC33-ECD-his his-tagged human LRRC33 protein extracellular domain fusion protein
  • hLTBP1-ECD-his his-tagged human LTBP1 protein extracellular domain fusion protein
  • control antibodies used in the present disclosure are all derived from published patent sequences. Unless otherwise specified, ARGX-115, SRK-Ab6, SRK181 and 28G11 control antibodies were recombinantly expressed using human IgG4+ ⁇ subtype. The h151D control antibody was recombinantly expressed using human IgG1+ ⁇ isotype. As an exception, the control antibodies used in the identification of the murine antibodies described in Example 3 were recombinantly expressed using the mouse mIgG2a+ ⁇ subtype ( Figure 6- Figure 9). The chimeric and humanized antibodies of the murine monoclonal antibody described in Example 3 of the present disclosure are all recombinantly expressed using human IgG4+ ⁇ subtype. All published patents in Table 1 are incorporated by reference into the present disclosure.
  • the expression and purification process of the antibody is as follows: the antibody sequence gene is synthesized and cloned into the expression vector pTT5, and then transiently transfected into HEK293 cells. After 7 days of shaking at 37°C, the cell supernatant is collected for protein A antibody purification. For the purification process, see "1.3" .2 Purification of Hybridoma Supernatant/Chimeric Antibody/Humanized Antibody by Protein A Affinity Chromatography”. The specific source and sequence information of the antibody are shown in Table 1.
  • the purification is carried out as follows: high-speed centrifugation of the cell expression supernatant sample to remove impurities. Equilibrate the nickel column with 20 mM PBS + 500 mM NaCl solution and rinse 2-5 column volumes. The supernatant sample is combined on the column, and the medium can choose nickel columns from different companies.
  • the collected eluted product containing the target protein can be further purified by gel chromatography Superdex200 (GE) after concentration.
  • the mobile phase is PBS to remove aggregates and impurity protein peaks, and collect the target product eluted peaks.
  • the obtained protein was identified as correct by electrophoresis, peptide map and LC-MS.
  • the proteins purified by this protocol include human GARP-His, human proTGF ⁇ 1-his, human GARP/TGF ⁇ 1 complex, cynomolgus monkey GARP/TGF ⁇ 1 complex, human LRRC33/TGF ⁇ 1 complex, and human LTBP1/TGF ⁇ 1 complex.
  • the cell culture supernatant expressing the antibody is first collected by high-speed centrifugation.
  • the Protein A affinity column was washed with 0.1M NaOH for 3-5 column volumes, and then washed with pure water for 3-5 column volumes.
  • the column was equilibrated for 3-5 column volumes using 1 ⁇ PBS (pH7.4) buffer system as equilibration buffer.
  • the cell supernatant was loaded and combined at a low flow rate, and the flow rate was controlled so that the retention time was about 1 min or longer. After the combination, the column was washed with 1 ⁇ PBS (pH 7.4) for 3-5 times the column volume until the UV absorption returned to the baseline.
  • solution replacement can be carried out by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or molecular exclusion such as G-25 desalting to replace the desired buffer system. Buffer system, or use a high-resolution size exclusion column such as Superdex 200 to remove the polymer component in the eluted product to improve the purity of the sample.
  • the nucleotide sequence encoding the full-length amino acid sequence of human GARP (UniProt number: Q14392) was cloned into pcDNA3.1-hygromycin vector (purchased from Clontech) and a plasmid was prepared, which was named as the plasmid encoding the full-length human GARP.
  • the nucleotide sequence encoding human full-length proTGF ⁇ 1 (UniProt number: P01137) was cloned into pcDNA3.1-puromycin vector (purchased from Clontech) and a plasmid was prepared and named as the human TGF ⁇ 1 encoding full-length plasmid.
  • HEK-293T cell line (purchased from ATCC) was transfected with a full-length plasmid encoding human GARP ( 3000 Transfection Kit, purchased from Invitrogen, catalog number: L3000-015), selectively cultured in DMEM medium containing 0.5mg/ml Hygromycin, 10% (w/w) fetal bovine serum for 2 weeks, with PE anti-human GARP Antibody (BioLegend, Cat. No. 352504) positive monoclonal cells were sorted into 96-well plates on a flow cytometer FACSAriaIII (purchased from BD Biosciences) and incubated at 37°C in 5% (v/v) CO for approx.
  • human GARP 3000 Transfection Kit, purchased from Invitrogen, catalog number: L3000-015
  • PE anti-human GARP Antibody BioLegend, Cat. No. 352504
  • the amplified clones were screened by flow cytometry.
  • the cell line with better growth, higher fluorescence intensity and monoclonal cell line was selected to continue to expand the culture and cryopreserved in liquid nitrogen for future use.
  • the obtained cell line was named hGARP-293T ( Figure 1).
  • the full-length plasmid encoding human GARP and the full-length plasmid encoding human TGF ⁇ 1 were co-transfected into HEK293T cells. After hygromycin and puromycin pressure screening, monoclonal selection, and ARGX-115 antibody for FACS identification, the resulting cell line was named hGARP complex -293T ( Figure 2). The same method was used to stably transfect CHOK1/S cells, and the resulting cell line was named hGARP-complex CHOK1/S. The expression of cell lines detected by FACS is shown in Figure 3.
  • the full-length amino acid sequence encoding the human integrin subunit ITGAV (Uniprot: P06756) was cloned into pcDNA3.1-hygromycin vector (purchased from Clontech) and plasmids were prepared.
  • the full-length amino acid sequence encoding the human integrin subunit ITGB6 (Uniprot: P18564) was cloned into the pcDNA3.1-Neo vector and a plasmid was prepared. Two plasmids, ITGAV and ITGB6, were used After simultaneous transient transfection of HEK293T cells with 3000 Transfection Kit (purchased from Invitrogen, Cat.
  • the monoclonal cell line with better growth and higher fluorescence intensity was selected to continue to expand the culture and cryopreserved in liquid nitrogen for future use.
  • the obtained cell line was named integrin ⁇ V ⁇ 6-293T.
  • Figure 4 shows the expression of cell lines detected by FACS.
  • the monoclonal antibodies of the present disclosure are produced by immunizing mice.
  • the mice used in the experiments were 6-8 weeks old, female SPF grade Balb/C mice or SJL mice (purchased from Charles River).
  • the immunogen was human GARP/TGF ⁇ 1 complex protein prepared in Example 1 or hGARP-complex CHOK1/S stably transfected cell line.
  • the immunogen was emulsified with TiterMax (purchased from Sigma, T2684-1M) and then injected subcutaneously (SC) and intraperitoneally (IP) with 0.1 mL, that is, 50 ⁇ g of immunogen was injected into each mouse;
  • the original ImjectAlum purchased from Thermo was injected subcutaneously and intraperitoneally with 0.1 ml, ie, 25 ⁇ g of immunogen was injected into each mouse.
  • 0.1 mL of TiterMax was emulsified with normal saline, and 0.1 mL of cell suspension was injected intraperitoneally 15 minutes later, that is, 1 ⁇ 10 7 cells were injected into each mouse; for booster immunization, the amount of cells injected intraperitoneally was 1 ⁇ 10 7 of cell suspension.
  • the frequency of immunization was once a week, and blood was drawn on the 3rd, 19th, 47th and 61st days.
  • Anti-human GARP/TGF ⁇ 1 monoclonal antibody can also be produced by immunizing rats, and the rats used in the experiment are 6-8 weeks old SD rats, purchased from Shanghai Slack. Human GARP/TGF ⁇ 1 complex protein was used as the immunogen, and the dose of immunogen was doubled compared with that of mice, and the first dose was 100 ⁇ g.
  • the immunization method and fusion screening method are the same as those of mice.
  • the Rab171 antibody in the present disclosure is obtained by immunizing rats.
  • the spleen and lymph nodes were taken aseptically, ground and filtered with a 40 ⁇ m cell strainer (purchased from BD Falcon), ACK Lysing Buffer (purchased from Gibco) was added, and the erythrocytes mixed in the splenocytes were lysed to obtain a spleen cell suspension, which was washed with DMEM (purchased from Gibco). Cells were washed once from Gibco's basal medium by centrifugation at 1500 rpm.
  • mice myeloma cells SP2/0 purchased from ATCC
  • Cytofusion Medium C purchased from BTX
  • the BTX ECM2001+ electrofusion method was used (refer to ECM2001+ELECTROFUSION PROTOCOL ) for cell fusion.
  • the fused cells were diluted into DMEM medium containing 20% fetal bovine serum (purchased from ExCell Bio) and 1 ⁇ HAT (purchased from Sigma). Splenocytes were then added to a 96-well cell culture plate at 2 ⁇ 10 4 per well and placed in a 37°C, 5% CO 2 incubator.
  • the supernatant of the cell fusion plate was screened by ELISA, and the positive clones detected by ELISA were expanded to a 24-well plate for expansion culture. After culturing for 3 days, the culture medium in the 24-well plate was taken, and the binding activity to human GARP/TGF ⁇ 1 complex protein was determined by ELISA and FACS.
  • the eligible hybridoma cells were selected for subcloning in the 6-well plate with Medium D (purchased from STEMCELL). 7 days after subcloning, single clones were picked and cultured in DMEM medium of 10% FBS and 1 ⁇ HT (purchased from Sigma) for 2 days at 37°C under 5% CO 2 conditions, and positive single clones were selected and expanded to 24 The plates were cultured continuously, and ELISA and FACS were used to determine the positive screening criteria for antigen binding after 2 days. According to the test results of the 24-well plate samples, the optimal clone was selected, and the optimal clone was expanded in DMEM medium containing 10% FBS at 37°C, 5% CO 2 , and frozen in liquid nitrogen. That is, the hybridoma cells of the present disclosure are obtained.
  • the supernatant of hybridoma cells was taken, and the binding to human GARP/TGF ⁇ 1 complex protein was detected by protein ELISA, and positive clones were selected; Binding of cells, human GARP-his protein, human proTGF ⁇ 1 protein, human GARP complex-293T cells.
  • the clones with higher positive signals in the initial screening were selected for subcloning, and the hybridoma antibodies after subcloning were detected by the same method.
  • the positive clones were classified according to their binding status, and there were 3 categories: (1) clones that only bound to GARP/TGF ⁇ 1 complex, but did not bind to GARP or proTGF ⁇ 1; (2) that bound to GARP/TGF ⁇ 1 complex and proTGF ⁇ 1, but not to GARP/TGF ⁇ 1 complex GARP-binding clones; (3) Clones that bind to GARP/TGF ⁇ 1 complex and GARP monomer, but not to proTGF ⁇ 1. Finally, 201 strains of hybridoma cells were obtained through screening and identification, and all of the 201 strains of antibodies could recognize the human GARP/TGF ⁇ 1 complex.
  • the 6 hybridomas with the strongest binding ability were selected from the 201 hybridoma clones after colonization for expanded culture (Mab017, Mab087, Mab138, Rab171, Mab195, Mab201), and the supernatant was collected and centrifuged according to Example 1.3.2
  • the purification method purifies the antibody.
  • the process of cloning antibody gene sequences from positive hybridoma cells is as follows: collect hybridoma cells in logarithmic growth phase, use Trizol (Invitrogen, Cat No. 15596-018) to extract RNA according to the kit instructions, and use PrimeScript TM Reverse Transcriptase kit to reverse Transcription (Takara, Cat No. 2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sent to a sequencing company for sequencing.
  • the murine antibodies Mab017, Mab087, Mab138, Rab171, Mab195 and Mab201 were obtained by sequencing, and their heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequences are shown below.
  • Bioinformatics methods were used to analyze the CDR regions of the above-mentioned antibodies, specifically through the Kabat numbering system, the Chothia numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and the IMGT numbering system (http:/ /www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) , the specific results are shown in Table 2.
  • VH/VL gene fragment was then subjected to homologous recombination with the expression vector pTT5 (with a signal peptide and human IgG4 constant region/ ⁇ constant region gene (CH1-Fc/CL) fragment) to construct a recombinant chimeric antibody full-length expression plasmid pTT5- VH-CH1-Fc/pTT5-VL-CL formed six chimeric antibodies ch-Mab017, ch-Mab087, ch-Mab138, ch-Rab171, ch-Mab195 and ch-Mab201.
  • the amino acid sequences of human IgG4 constant region and kappa constant region are shown in Table 1.
  • the antibody heavy chain and light chain were transfected into Expi293F cells according to the plasmid ratio of 1:1. After 7 days of shaking at 37°C, the supernatant was collected, centrifuged, and the antibody was purified according to the purification method described in Example 1.3.2. .
  • IMGT http://imgt.cines.fr
  • MOE Molecular Operating Environment, molecular operating environment
  • the heavy and light chain variable region germline genes of the murine antibody are used as templates, and the CDRs of the murine antibody (the CDRs determined by the Kabat numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)) They were transplanted into corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antibody sequence of this example is numbered according to the Kabat numbering system and its CDR regions are determined . Unless otherwise specified, in the antibody sequences of this example, italics indicate framework regions, bold fonts + underlines indicate CDR regions, and bold fonts + character borders indicate back mutations or hotspot mutations.
  • the humanized light chain templates of the murine antibody Mab017 are IGKV1-39*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01, and the CDRs of the murine antibody Mab017 were transplanted into them respectively.
  • the humanized antibody h017 of Mab017 was obtained, and its variable region sequence is shown below.
  • the key amino acids in the FR region sequence of the Mab017 humanized antibody were backmutated to ensure the original affinity.
  • the amino acid mutation of NG was performed to eliminate the molecule
  • the modification risk, the specific mutation design is shown in Table 3.
  • Graft represents the insertion of the mouse antibody CDR into the human germline FR region sequence
  • Graft+A43S represents the mutation of A at position 43 of Graft to S, and so on.
  • h017H1L1 indicates that the Mab017 humanized antibody h017 has a light chain variable region as described in h017L1 and a heavy chain variable region as described in h017H1, and so on.
  • amino acid sequences of h017L1, h017L2, h017L3, h017L4, h017H1, h017H2, h017H2a, h017H2b, h017H2c, h017H2d, h017H2e, h017H3 and h017H4 are shown below.
  • h017L1 (same as h017 LCVR) (SEQ ID NO: 28):
  • the humanized light chain templates of the murine antibody Mab087 are IGKV4-1*01, IGKV2D-29*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV2-26*01 and IGHJ6*01.
  • the murine antibody Mab087 The CDRs were grafted into their human templates, that is, the humanized antibody h087 of Mab087 was obtained.
  • the h087 variable region sequence is shown below.
  • Graft represents the insertion of mouse antibody CDRs into the human germline FR region sequence
  • A93G represents the mutation of A to G at position 93 of Graft, and so on.
  • h087H1L1 indicates that the Mab087 humanized antibody h087 has a light chain variable region as described in h087L1 and a heavy chain variable region as described in h087H1, and so on.
  • the humanized light chain templates of the murine antibody Mab138 are IGKV1-39*01, IGKV4-1*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV3-7*01, IGHV3-30*02, IGHV3-15 *01 and IGHJ6*01, the CDRs of the murine antibody Mab138 were grafted into its human template, respectively, to obtain the humanized antibody H138 of Mab138.
  • the variable region sequence is as follows.
  • H138 LCVR1 (VL-CDR graft1, IGKV1-39*01) (SEQ ID NO: 52):
  • H138 LCVR2 (VL-CDR graft2, IGKV4-1*01) (SEQ ID NO: 53):
  • Graft represents the insertion of mouse antibody CDRs into the human germline FR region sequence
  • L46A represents the mutation of the 46th L of Graft to A, and so on.
  • h138H1L1 indicates that the Mab138 humanized antibody h138 has a light chain variable region as described in h138H1 and a heavy chain variable region as described in h138L1, and so on.
  • h138H1, h138H2, h138H3, h138H4, h138H5, h138H6, h138H7, h138H8, h138L1, h138L2, h138L3, h138L4, h138L5, h138L6 are shown below.
  • h138H6 (SEQ ID NO: 59):
  • the humanized light chain templates of the mouse antibody Rab171 are IGKV1-39*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV3-30*01 and IGHJ3*01, and the CDRs of the mouse antibody Rab171 were transplanted into them respectively.
  • the humanized template the humanized antibody h171 of Rab171 was obtained, and its variable region sequence was as follows.
  • h171 HCVR VH-CDR graft, IGHV3-30*01 (SEQ ID NO: 68):
  • H171 LCVR (VL-CDR graft, IGKV1-39*01) (SEQ ID NO: 69):
  • the key amino acids in the FR region sequence of the humanized antibody of Rab171 were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity; at the same time, based on the existence of high-risk easy-to-modify site NG in the CDR2 region of the light chain of Rab171 , so the amino acid mutation of NG was carried out according to the antibody structure by computer simulation to eliminate the risk of molecular modification.
  • the specific mutation design is shown in Table 9.
  • Graft represents the insertion of mouse antibody CDRs into the human germline FR region sequence
  • A43S represents the mutation of A at position 43 of Graft to S, and so on.
  • the Rab171 humanized antibody back mutation and hotspot mutation design in Table 9 were combined to finally obtain a variety of Rab171 humanized antibodies (see Table 10 for details).
  • h171H1L1 indicates that the Rab171 humanized antibody h171 has a light chain variable region as described in h171L1 and a heavy chain variable region as described in h171H1, and so on.
  • the humanized light chain templates of the murine antibody Mab195 are IGKV1-NL1*01, IGKV2-28*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-18*01 and IGHJ6*01.
  • the murine antibody Mab195 The CDRs of 1 were grafted into its human template, namely, the humanized antibody h195 of Mab195 was obtained, and its variable region sequence was as follows.
  • h195 HCVR also known as VH-CDR graft, IGHV1-18*01 (SEQ ID NO: 78):
  • h195 LCVR1 also known as VL-CDR graft1, IGKV1-NL1*01 (SEQ ID NO: 79):
  • h195 LCVR2 also known as VL-CDR graft2, IGKV2-28*01 (SEQ ID NO: 80):
  • the key amino acids in the FR region sequence of the humanized antibody of Mab195 were back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity; at the same time, based on the existence of a high-risk easy-to-modify site NG in the CDR2 region of the Mab195 heavy chain , so the amino acid mutation of NG was carried out according to the antibody structure by computer simulation to eliminate the risk of molecular modification.
  • the specific mutation design is shown in Table 11.
  • Graft represents the insertion of mouse antibody CDRs into the human germline FR region sequence
  • A43S represents the mutation of A at position 43 of Graft to S, and so on.
  • h195H1L1 indicates that the Mab195 humanized antibody h195 has a light chain variable region as described in h195L1 and a heavy chain variable region as described in h195H1, and so on.
  • the humanized light chain templates of the murine antibody Mab201 are IGKV3-11*01, IGKV6-21*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-8*01 and IGHJ6*01.
  • the murine antibody Mab201 The CDRs of H201 were grafted into its human template, that is, the humanized antibody h201 of Mab201 was obtained, and its variable region sequence was as follows.
  • h201 LCVR1 (VL-CDR graft1, IGKV3-11*01) (SEQ ID NO: 91):
  • Graft represents the insertion of mouse antibody CDRs into the human germline FR region sequence
  • L48W represents the mutation of the 48th L of Graft to W, and so on.
  • h201H1L1 indicates that the Mab201 humanized antibody h201 has a light chain variable region as described in h201L1 and a heavy chain variable region as described in h201H1, and so on.
  • h201H1, h201H2, h201H3, h201H4, h201H5, h201H6, h201H7, h201L1, h201L2, h201L3, h201L4, h201L5 are shown below.
  • h201L4 (SEQ ID NO: 103):
  • the coding gene fragments of VH/VL of each humanized antibody in Example 6 were homologously recombined with the expression vector pTT5-huIgG4HC or PTT5-huIgGLC (Kappa) (with signal peptide and constant region gene) by gene synthesis, and the whole antibody was constructed. long expression vector. See Table 1 for the heavy and light chain constant region amino acid sequences.
  • the constructed antibody heavy chain and light chain plasmids were co-transfected into Expi293F cells at a ratio of 1:1 respectively. After culturing for 7 days in a 37°C incubator with a shaker at 120 rpm, collect the cell supernatant, centrifuge and follow the steps of 1.3.1. Antibodies were purified using protein A.
  • the BIAcore 8K instrument was used to detect the binding strength of antibody and antigen by anti-mouse antibody capture method.
  • the Anti-Mouse IgG antibody was immobilized on the CM5 chip by amino coupling method, with HBS-EP+pH7.4 as the mobile phase, after mixing NHS and EDC, activated
  • the Anti-Mouse IgG antibody was diluted to 15 ⁇ g/mL with 10 mM sodium acetate pH5.0, injected for 420 seconds, and finally the remaining activated sites were blocked with ethanolamine.
  • the affinity of the antibody to the antigen was determined by multi-cycle kinetics method.
  • the antibody to be tested was first captured with an anti-mouse antibody, and then a single concentration of human GARP/TGF ⁇ 1 protein complex antigen (prepared by Example 1) was injected. ), record the binding and dissociation process of antibody and antigen protein, and finally complete chip regeneration with Glycine pH1.7, wherein the mobile phase is HBS-EP+pH7.4 (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05%surfactant P20), The flow rate was 30 ⁇ L/min, the regeneration time was 30 seconds, and the detection temperature was 25°C. Finally, according to the 1:1 binding model, the data were analyzed, the kinetic parameters of antibody-antigen binding were fitted, and the equilibrium dissociation constant KD was obtained. The specific test results are shown in Table 16.
  • the BIAcore 8K instrument was used to detect the binding strength of antibody and antigen by Protein A capture method.
  • protein A was immobilized on the CM4 chip by amino coupling method. According to the instructions of the Amine Coupling Kit, HBS-EP+pH7.4 was used as the mobile phase. After mixing NHS and EDC, the chip was activated for about 600 seconds. Protein A was diluted to 50 ⁇ g/mL with 10 mM sodium acetate pH 4.5, injected for 600 s, and finally the remaining activated sites were blocked with ethanolamine. Then, the affinity of the antibody to the antigen was determined by multi-cycle kinetics method.
  • the chimeric antibody to be tested (Example 5) or the humanized antibody (Example 7) to be tested was first captured with a Protein A chip, and then injected Single concentration of human GARP/TGF ⁇ 1 protein complex or cynomolgus monkey GARP/TGF ⁇ 1 protein complex or human proTGF ⁇ 1 monomer antigen (prepared by Example 1), record the binding and dissociation process of antibody and antigen protein, and finally use Glycine pH1. 5.
  • Affinity with human GARP/TGF ⁇ 1 protein 017 humanized antibody combination (Table 4) affinity is less than 7.11E-09M; 087 humanized antibody combination (Table 6) affinity is less than 3.38E-11M; 138 humanized combination (Table 8) All affinities are less than 9.07E-09M; 171 humanized antibody combinations (Table 10) all have affinities less than 8.07E-09M; 201 humanized antibody combinations (Table 14) all have affinities less than 1.66E-09M; 195 human The affinities of the combined antibody combinations (Table 12) were all less than 5.69E-10M.
  • the humanized combination that can maintain the affinity of the chimeric antibody or the affinity drop within 2-fold was selected as the final molecule, and its affinity with the cynomolgus monkey GARP/TGF ⁇ 1 protein complex or with the human proTGF ⁇ 1 monomer was tested (see Table for details). 17).
  • Example 9 Enzyme-linked immunosorbent assay to detect the binding of antibodies to human GARP protein, proTGF ⁇ 1 protein, GARP/TGF ⁇ 1 protein complex, LTBP1/TGF ⁇ 1 protein complex, LRRC33/TGF ⁇ 1 protein complex
  • the six antibody molecules of the present disclosure have different binding properties to GARP protein, proTGF ⁇ 1 protein and GARP/TGF ⁇ 1 complex.
  • the ELISA detection method used in the present disclosure is as follows: the antigen protein (prepared by Example 1) was diluted with pH 7.4 PBS to 4 ⁇ g/mL, added to an ELISA plate (corning, CAT#9018), 50 ⁇ L/well, 4°C overnight , shake off the coating solution, add 5% non-fat milk powder (Biotek, CAT#A600669-0250)-PBS, 250 ⁇ L/well, incubate at 37°C for 2-4 hours, on a plate washer (Biotek, CAT#405TUS) Wash three times with 0.05% Tween20-PBS (Sangon Bio, CAT#A100777-0500, B548117-0500), and add the dilution of purified antibody (purified mouse antibody, human-mouse chimeric antibody, and humanized antibody are all used 1% BSA diluted
  • Mab087 can bind to human proTGF ⁇ 1 monomer and the complex formed by proTGF ⁇ 1 (human GARP/TGF ⁇ 1 complex, human LTBP1/TGF ⁇ 1 complex), but not to GARP-His monomer.
  • proTGF ⁇ 1 human GARP/TGF ⁇ 1 complex, human LTBP1/TGF ⁇ 1 complex
  • Such antibodies have similar binding properties to the control antibody 28G11, ie the binding site is on the proTGF ⁇ 1 monomer.
  • FIGS. 8A to 8D The binding of murine antibody Mab138 to human GARP, proTGF ⁇ 1, GARP/TGF ⁇ 1 complex, and LTBP1/TGF ⁇ 1 complex is shown in FIGS. 8A to 8D .
  • Mab138 neither binds to GARP-his nor proTGF ⁇ 1 protein, but only binds to the complex formed by GARP/TGF ⁇ 1, that is, the binding site is on both GARP and proTGF ⁇ 1.
  • the binding properties of this antibody are similar to the control antibody ARGX-115.
  • Rab171 does not bind to GARP monomer nor proTGF ⁇ 1 monomer, but only to the complex formed by GARP/TGF ⁇ 1.
  • the binding properties of the antibody are similar to Mab138 and control antibody ARGX-115, and the binding sites are both on GARP and proTGF ⁇ 1.
  • Mab195 is known to bind to GARP and the GARP/TGF ⁇ 1 complex, but not to proTGF ⁇ 1 (specific quantitative data not shown).
  • the binding ability of the modified humanized antibody to human GARP, proTGF ⁇ 1, GARP/TGF ⁇ 1 complex, LTBP1/TGF ⁇ 1 and LRRC33/TGF ⁇ 1 complex was further detected by ELISA.
  • the detection method of humanized antibody is the same as that of mouse antibody, and the use of secondary antibody is changed from anti-mouse to anti-human.
  • the binding ability of the humanized antibody h087-H2L2 to the LRRC33/TGF ⁇ 1 complex was detected by ELISA.
  • h087-H2L2 can well bind the complex formed by LRRC33/TGF ⁇ 1.
  • Example 10 ELISA detects the binding of antibody to cynomolgus monkey GARP/TGF ⁇ 1 complex
  • Humanized antibodies h017-H2dL2, h087-H2L2, h138-H8L5, h171-H3L5, h195-H3L1, h201-H5L3 and control antibodies were analyzed with cynomolgus monkey GARP/TGF ⁇ 1 according to the ELISA assay described in Example 9 Binding of protein complexes (prepared from Example 1). As shown in Figure 17 and Table 18, the aforementioned antibodies have good cross-binding activity with cynomolgus monkey GARP/TGF ⁇ 1 complex.
  • N/A means poor fit or invalid fit
  • Example 11 FACS detects the binding activity of antibody to cells expressing cynomolgus monkey GARP/TGF- ⁇ 1 complex
  • 293T cells were collected, 5 ⁇ 10 6 cells were seeded into a 15cm cell culture dish (corning, CAT#430599), cultured overnight at 37°C 5% CO 2 , and the next day was replaced with fresh 10% FBS-DMEM medium (Excell, CAT# FSP500; Gibco, CAT#11995), according to the instructions of Liposome 3000 transfection kit (invitrogen, L3000-015), the cynomolgus monkey GARP plasmid and cynomolgus monkey proTGF ⁇ 1 plasmid were transiently expressed.
  • h017-H2dL2, h087-H2L2, h138-H8L5, h171-H3L5, h195-H3L1, h201-H5L3 could well bind the cynomolgus GAPR/TGF ⁇ 1 complex at the cellular level.
  • Antibody name Emax EC50, nM ARGX-115-hIgG4 14948 0.3808 GARP-h138-H8L5 14487 0.9342 GARP-h201-H5L3 14196 0.7067 GARP-h171-H3L5 13461 0.4443 GARP-h017-H2dL2 17592 0.3117 GARP-h195-H3L1 20173 0.3526 GARP-h087-H2L2 23180 0.429 SRK-Ab6-hIgG4 19376 0.3298 28G11-hIgG4 16949 0.4524 Anti-Hel-hIgG4 339 N/A
  • N/A means poor fit or no fit
  • Example 12 FACS detects the binding of antibodies to overexpressing human GARP-293T cells and GARPcomplex-293T cells
  • the overexpression cell line constructed in Example 2 was used. Cells were collected, washed once with PBS (Hyclone, CAT#SH30256), resuspended in 1% BSA-PBS to 2 ⁇ 10 5 /50 ⁇ L, diluted with 1% BSA-PBS to 270 nM, and serially diluted 3 times at 12 concentration points.
  • PBS Hyclone, CAT#SH30256
  • Binding of humanized antibodies h017-H2dL2, h087-H2L2, h138-H8L5, h171-H3L5, h201-H5L3, h195-H3L1 to human GARP-293T overexpressing cells is shown in Figure 19A and Table 20. Among them, h017-H2dL2 and h195-H3L1 antibodies can well bind GARP monomers at the cellular level.
  • Antibody name Emax EC50, nM ARGX-115-hIgG4 629 N/A GARP-h138-H8L5 477 N/A GARP-h201-H5L3 413 N/A GARP-h171-H3L5 1205 N/A GARP-h017-H2dL2 22211 0.3456 GARP-h195-H3L1 28574 0.217 GARP-h087-H2L2 691 N/A SRK-Ab6-hIgG4 521 N/A 28G11-hIgG4 123 N/A Anti-Hel-hIgG4 105 N/A
  • N/A means no fit or poor fit
  • Antibody name Emax EC50, nM ARGX-115-hIgG4 31578 0.5955 GARP-h138-H8L5 30743 1.251 GARP-h201-H5L3 26951 0.9619 GARP-h171-H3L5 30622 0.7121 GARP-h017-H2dL2 34050 0.4934 GARP-h195-H3L1 35114 0.4681 GARP-h087-H2L2 38998 0.3435 SRK-Ab6-hIgG4 35249 0.3845 28G11-hIgG4 29123 0.4173 Anti-Hel-hIgG4 37.2 N/A
  • N/A means no fit or poor fit
  • Human Treg cells were isolated from human PBMCs using a sorting kit (Stemcell, Cat. No.: 18063), stimulated and expanded in vitro with Dynabeads Human Treg Expander (Gibco, Cat. No.: 11129D) for 15 days, and then were obtained in aliquots and frozen.
  • the Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300 ⁇ g for 5 minutes the next day, resuspended in DPBS to obtain a cell suspension, and counted.
  • the number of cells required for the experiment was added to a centrifuge tube, centrifuged at 300 ⁇ g for 5 min, the supernatant was removed, and the cell density was adjusted to 2 ⁇ 10 6 /ml with staining buffer (2% FBS in PBS v/v), and plated with 96 Well plate, 50 ⁇ l per well. Take all test antibodies and control antibody anti-Hel isotype (diluted with staining buffer to the highest concentration of 10 ⁇ g/ml, and then diluted 5 times, a total of 8 concentrations). Test and control antibodies were added to each well, 50 ⁇ l per well. Place the well plate with the cell suspension and the antibody on a microplate shaker, shake at 500 rpm for 1 min, and mix the cells and the antibody well.
  • the plate was placed in a 4°C refrigerator and incubated for 30 min. After incubation, add 100 ⁇ l of staining buffer to each well, centrifuge at 350 ⁇ g for 5 min, and discard the supernatant; add 200 ⁇ l of staining buffer to each well to resuspend the cells, centrifuge at 350 ⁇ g for 5 min, and discard the supernatant. Add the staining buffer to PE goat anti-Human IgG Fc (eBioscience, product number: 12-4998-82) at a ratio of 250:1 (staining buffer: corresponding staining fluorescent antibody) to prepare a staining solution, and add to the cells after mixing.
  • PE goat anti-Human IgG Fc eBioscience, product number: 12-4998-82
  • h017-H2dL2, h087-H2L2, h138-H8L5, h171-H3L5, h195-H3L1 and h201-H5L3 could effectively bind to Treg cells.
  • the fluorescence intensity of antibody binding to Treg cells is shown in Table 22.
  • the experimental procedure employed in this disclosure is as follows: On the first day, medium (MEM, Gibco, 11095, +0.5% FBS, 1% non-essential amino acids, Gibco, 11140, 1 mM sodium pyruvate, 1% penicillin , Gibco, 15140,) diluted the purified antibody samples, 3-fold dilution from 50nM to 9 concentration points; collected hGARP-complex-293T stable transfected cells and ⁇ 6-293T stable transfected cells, and resuspended in the medium; The suspended cells were sequentially added to a 96-well cell culture plate (corning, CAT#3799), and cultured at 37°C and 5% CO 2 for 24 hours.
  • medium Gibco, 11095, +0.5% FBS, 1% non-essential amino acids, Gibco, 11140, 1 mM sodium pyruvate, 1% penicillin , Gibco, 15140,
  • the suspended cells were sequentially added to a 96-well cell culture plate (corning,
  • TGF ⁇ /SMAD Signaling Pathway SBE Reporter-HEK293 TGF ⁇ /SMAD Signaling Pathway SBE Reporter-HEK293 Cell Line, BPS, CAT#60653
  • MEM recovery medium
  • FBS+1% non-essential amino acids Gibco, 11140, + 1 mM non-essential amino acids, Gibco, 11140, + 1% penicillin-streptomycin, Gibco, 15140
  • 96-well cell culture plates corning, CAT#3599
  • 100 ⁇ L/well 100 ⁇ L/well
  • the intensity of fluorescein signal can represent the secretion of active TGF ⁇ 1. It can be found from the results ( FIG. 22 , Table 23 ) that the antibodies h017-H2dL2, h087-H2L2, h138-H8L5, h171-H3L5, h201-H5L3, h195-H3L1 antibodies in the present disclosure all significantly inhibit the secretion of activated TGF ⁇ 1 .
  • Antibody IC50,nM ARGX-115 0.5103 h138-H8L5 0.6907 h201-H5L3 0.5220 h171-H3L5 0.5486 h017-H2dL2 0.4630 h195-H3L1 0.6033 h087-H2L2 0.8371 SRK-Ab6-hIgG4 0.5480 28G11-hIgG4 1.134 Anti-HelhIgG4 N/A
  • N/A means "no fit or poor fit
  • Example 16 Anti-human GARP/TGF ⁇ 1 antibody inhibits the production of active TGF ⁇ 1 factor by human Treg cells
  • p-SMAD2 forms a complex with pSMAD3 and SMAD4, and enters the nucleus to regulate the transcription of a series of downstream genes.
  • the phosphorylation level of p-SMAD2 can represent the activation degree of the signaling pathway, which in turn reflects the amount of active TGF ⁇ 1 factor produced by human Treg cells.
  • Human Treg cells were isolated from human PBMCs using a sorting kit (Stemcell, Cat. No.: 18063), and were obtained after 15 days of in vitro stimulation and expansion with Dynabeads Human Treg Expander (Gibco, Cat. No. 11129D) and frozen in aliquots.
  • the Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300 ⁇ g for 5 minutes the next day, and resuspended in AIM-V medium (Gibco, Cat. No. 31035025) to obtain a cell suspension and counted.
  • a positive control well was set, and 20 ⁇ l/well hTGF- ⁇ 1 (Peprotech, product number: 100-21) was added to the positive control well (diluted to 60ng/ml with AIM-V, the final concentration was 10ng/ml), and the Treg only well was set as Treg cells without drug and Dynabeads were used as negative controls.
  • BD Phosflow TM Fix Buffer I (BD, catalog number: 557870) pre-warmed at 37°C, and fix at 37°C for 10 min. The cells were washed twice with Incubation buffer, the centrifugation speed was increased to 400g for 4 min, and the supernatant was discarded. Then slowly add 100 ⁇ l/well of pre-cooled BD Phosflow TM Perm Buffer III (BD, Cat. No.: 558050) to resuspend the cells, and react on ice for 30 min.
  • BD Phosflow TM Fix Buffer I (BD, catalog number: 557870) pre-warmed at 37°C, and fix at 37°C for 10 min. The cells were washed twice with Incubation buffer, the centrifugation speed was increased to 400g for 4 min, and the supernatant was discarded. Then slowly add 100 ⁇ l/well of pre-cooled BD Phosflow TM Perm Buffer III (BD, Cat. No.: 558050) to
  • the antibodies h017-H2dL2, h087-H2L2, h138-H8L5, h171-H3L5, h195-H3L1 and h201-H5L3 of the present disclosure can effectively inhibit the phosphorylation of SMAD2 in Treg cells, indicating that they can effectively inhibit the phosphorylation of SMAD2 in Treg cells.
  • Human Treg cells produce active TGF ⁇ 1 factor.
  • Example 17 Anti-GARP/TGF ⁇ 1 antibody inhibits human Treg cell function in vivo
  • Treg is an important immune cell that regulates the rate and extent of GHVD, and if the suppressive ability of Treg on T cells is down-regulated, it will promote the occurrence of GVHD.
  • Treg cells were isolated from PBMCs of the same donor using Stem cell human CD4+CD127lowCD25+Treg isolation kit (cat#18063) and then Dynabeads (bead-to -cell ratio at 2:1) and hIL-2 (200IU/mL) and 1 ⁇ M Rapamycin were amplified in vitro for 7 days, after 7 days without adding Rapamycin, Treg cells were collected after 16 days of expansion and were amplified with fluorescent dye-coupled HuCD45, CD3 , CD4, CD25, FoxP3 antibody staining. Treg cell purity (>93% purity) was determined by flow cytometry and the expanded Treg cells were frozen for later use.
  • mice On day -1, the animals were randomly divided into groups according to their body weight and administered, with 10 animals in each group, once a week, for a total of 6 times, with a dose of 20 mg/kg. Then on day 0, expanded human Tregs ( 5 x 106 per mouse) mixed with human PBMCs ( 5 x 106 per mouse) were tail vein injected. On days 14 and 28, expanded human Tregs (2.5 x 10 ⁇ 6 > per mouse) were each re-injected into the tail vein. The occurrence of GVHD in mice was measured once a week for the first two weeks of the dosing and observation period, and twice a week for the next 4 weeks.
  • GVHD scores were established based on: weight loss (0 points: ⁇ 10%, 1 point: 10%-20%, 2 points: >20%, 3 points: >30%), anemia (0 points: red or pink Tail; 1 point: white tail), posture (0 point: normal, 1 point: hunched back), general activity (0 point: normal, 1 point: restricted), hair removal (0 point: no hair removal, 1 point: hair removal ) and jaundice (0 points: white or red tail, 1 point: yellow tail). Maximum disease severity or death corresponds to 7 points.
  • h138, h017 and h087 refer to the humanized full-length antibodies h138-H8L5, h017-H2dL2 and h087-H2L2 prepared according to Example 7, respectively.
  • FIGS. 24A to 24B and Table 24 For specific results, see FIGS. 24A to 24B and Table 24. As shown in Figure 24A, on the 36th day after the first administration, the overall GVHD score curve of the mice in the h017 group was higher than that in the PBS group, and the onset of GVHD (day 18) was significantly earlier than that in the PBS control group (day 29). At the same time, the GVHD score (5.63 points) of the h017 group on the 36th day was significantly higher than that of the PBS control group (1.88 points).
  • the GVHD score of the mice in the h087 administration group was significantly higher than that in the PBS control group (p ⁇ 0.001).
  • the GVHD score of the mice in the h138 administration group was higher than that in the control PBS group (p ⁇ 0.01).
  • Figure 24A shows that compared with the control antibody ARGX-115, the GVHD score of the mice in the h017 administration group was significantly higher than that in the ARGX-115 administration group (**, p ⁇ 0.01); the GVHD score of the mice in the h087 administration group was significantly higher than that in the ARGX-115 administration group (**, p ⁇ 0.01).
  • ARGX-115 administration group (****, p ⁇ 0.001).
  • mice of SPF grade Female wild-type C57 mice of SPF grade, 6-8 weeks old, weighing about 18-20g, 3 mice in each group, were given 10mg by a single tail vein injection Control antibodies (ARGX-115, SRK-Ab6) and 6 humanized antibodies (h138-H8L5, h017-H2dL2, h087-H2L2, h171-H3L5, h201-H5L3, h195- H3L1).
  • ARGX-115, SRK-Ab6 6 humanized antibodies
  • mice were fed with standard chow without food and water.
  • the drug is diluted with normal saline.
  • Orbital blood was collected at the time points before administration, 0.25 hours, 2 hours, 24 hours, 72 hours, 168 hours, 240 hours, 336 hours, 504 hours and 672 hours after administration.
  • the blood samples were collected in a micro blood collection tube and left standing for about 30 minutes, then centrifuged at 12000rpm for 5 minutes at 4°C, and the serum was separated into a low adsorption centrifuge tube, and the compound code and time point were marked.
  • mice Nine SPF-grade female human GARP knock-in C57 mice, 6 weeks old, weighing about 19-21 g, were divided into 3 groups, 3 mice in each group, and given a single tail vein injection with a dose of 10 mg/kg of the control antibody (ARGX -115) and the antibody to be tested (h138-H8L5 and h017-H2dL2 prepared in Example 7.2), and compare their pharmacokinetic differences.
  • ARGX -115 the control antibody
  • h138-H8L5 and h017-H2dL2 prepared in Example 7.2
  • mice were fed with standard chow without food and water.
  • the drug is diluted with normal saline.
  • Orbital blood was collected at the time points before administration, 0.25 hours, 2 hours, 24 hours, 72 hours, 168 hours, 240 hours, 336 hours, 504 hours and 672 hours after administration.
  • the blood samples were collected in a micro blood collection tube and left standing for about 30 minutes, then centrifuged at 12000rpm for 5 minutes at 4°C, and the serum was separated into a low adsorption centrifuge tube, and the compound code and time point were marked.

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Abstract

L'invention concerne un anticorps qui se lie de manière spécifique à un composé GARP/TGFβ1 ou à un fragment de liaison à l'antigène de celui-ci, une molécule de liaison à un antigène multispécifique et un récepteur antigénique chimérique le contenant, une cellule effectrice immunitaire exprimant le récepteur antigénique chimérique, et une composition pharmaceutique préparée à partir de l'anticorps, une utilisation pharmaceutique de l'anticorps, et une utilisation de l'anticorps dans le traitement d'une maladie. La présente invention concerne en outre un acide nucléique codant, un vecteur contenant l'acide nucléique, une cellule hôte contenant l'acide nucléique ou le vecteur, et un procédé de préparation et de purification de l'anticorps.
PCT/CN2022/083667 2021-03-29 2022-03-29 ANTICORPS GARP/TGFβ1 ET SON UTILISATION WO2022206753A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016125017A1 (fr) * 2015-02-03 2016-08-11 Universite Catholique De Louvain Protéine anti-garp et ses utilisations
CN109071646A (zh) * 2016-03-11 2018-12-21 供石公司 TGFβ1-结合免疫球蛋白及其用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016125017A1 (fr) * 2015-02-03 2016-08-11 Universite Catholique De Louvain Protéine anti-garp et ses utilisations
CN109071646A (zh) * 2016-03-11 2018-12-21 供石公司 TGFβ1-结合免疫球蛋白及其用途

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