CN117043187A

CN117043187A - GARP/TGF beta 1 antibodies and uses thereof

Info

Publication number: CN117043187A
Application number: CN202280022651.3A
Authority: CN
Inventors: 成广存; 葛虎; 曹卓晓; 唐任宏; 任晋生
Original assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Current assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Priority date: 2021-03-29
Filing date: 2022-03-29
Publication date: 2023-11-10
Also published as: WO2022206753A1

Abstract

The present disclosure relates to antibodies that specifically bind to GARP/tgfβ1 complex and uses thereof, and specifically discloses antibodies or antigen binding fragments, multi-characteristic antigen binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid molecules, vectors, cells, methods of preparation, pharmaceutical compositions, pharmaceutical uses, and methods of treatment of diseases that bind to GARP/tgfβ1 complex, are of great significance for the development of medicaments for the treatment of tgfβ -related diseases.

Description

GARP/TGF beta 1 antibodies and uses thereof

The present disclosure claims priority from the chinese patent office, application number 202110330064.8, chinese patent application entitled "GARP/tgfβ1 antibody and use thereof," filed on day 29, 3, 2021, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to the field of antibodies, in particular, GARP/tgfβ1 antibodies and uses thereof.

Background

Transforming growth factor beta 1 (tgfβ1) is a member of the tgfβ protein family. Tgfβ comprises three specific subtypes tgfβ1, tgfβ2 and tgfβ3, each subtype being encoded by a separate gene. Tgfβ was originally produced as a precursor (pro-TGF-b) in vivo and was converted to latent protein in cells by furin proprotein convertase. Latent tgfβ is composed of the N-terminal LAP protein and the C-terminal mature tgfβ in a non-covalent manner. Latent tgfβ cannot bind to receptors and activate downstream signaling pathways. The active form of TGF-beta exists as a homodimer free of LAP, with a molecular weight of about 25kd. Mature TGF-beta factors induce receptor phosphorylation and activation of downstream p-SMAD2/3 signaling pathways by binding to the receptors TβRI and TβRII.

The production of mature tgfβ is tightly regulated by multiple steps. A significant amount of tgfβ in the body is present in an inactive complex state, e.g., LTBP1/tgfβ complex, LTBP3/tgfβ complex, covalently bound to the latent transforming growth factor binding protein LTBP1 or LTBP 3. LTBP1 and LTBP3 are mainly secreted and expressed by fibroblasts, and a large amount of LTBP 1/TGF-beta complex and LTBP 3/TGF-beta complex exist in the extracellular matrix in a latent state. And then activated by proteases such as MMP2, MMP9 and plasmin to become mature TGF beta. Tgfβ can also exist in a membrane bound form, and membrane bound tgfβ complexes include two types: GARP/tgfβ and LRRC33/tgfβ complexes. Among them, GARP is mainly expressed in regulatory T cells (tregs), which are critical for regulating tgfβ activation. The GARP/tgfβ complex is displayed on Treg cell expression and then releases mature tgfβ under the action of integrins.

Tgfβ is a multifunctional cytokine that inhibits proliferation of many cell types, including epithelial cells, endothelial cells, hematopoietic cells, and immune cells. The data show that in humans, the tgfβ1 subtype predominates and that activation of tgfβ signaling pathways is mediated primarily by tgfβ1. Excessive activation of tgfβ and its signaling pathways will cause tgfβ -related diseases such as cancer, tumors, inflammation, fibrotic diseases and cardiovascular and cerebrovascular diseases.

Thus, targeting tgfβ1 is of great clinical value for the treatment of tgfβ1-related diseases. Studies have shown that fully neutralising antibodies to TGF-beta 1/2/3 do not produce anti-tumour effects. Small molecule inhibitors based on tgfp receptor-associated may produce heart-related toxicity, thereby limiting higher dose applications such as ALK5 inhibitors. The present disclosure provides antibody molecules that specifically bind to the GARP/tgfβ1 complex and/or monomers therein thereby blocking the maturation process of tgfβ1, reducing the release of active tgfβ1, and are of great significance in the treatment of tgfβ1-related diseases.

Disclosure of Invention

The present disclosure provides antibodies or antigen binding fragments that bind to GARP/tgfβ1 complexes, multi-property antigen binding molecules, chimeric antigen receptors, immune effector cells, nucleic acid molecules, vectors, cells, methods of manufacture, pharmaceutical compositions, pharmaceutical uses, and methods of treatment of diseases.

In a first aspect, the present disclosure provides an antibody or antigen binding fragment that binds to a GARP/tgfβ1 complex, the antibody or antigen binding fragment comprising HCDR1-3, and/or LCDR1-3, the HCDR1-3 and/or the LCDR1-3 comprising a sequence shown in table 2 or table 15, respectively, or a sequence having at least 70% identity or at most 5 mutations to a sequence shown in table 2 or table 15;

Preferably, 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, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the HCDR2 comprises SEQ ID NO: 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 thereto or at most 5 mutations;

preferably, the HCDR3 comprises SEQ ID NO: 106. 111, 119, 124, 132, 137, 145, 150, 158, 163, 171, or 176, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the LCDR1 comprises SEQ ID NO: 112. 115, 125, 128, 138, 141, 151, 154, 164, 167, 177 or 180, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the LCDR2 comprises a sequence corresponding to SEQ ID NO: 113. 116, 126, 129, 139, 142, 152, 187, 155, 165, 168, 178 or 181, or a sequence having at least 70% identity thereto or at most 5 mutations;

Preferably, the LCDR3 comprises SEQ ID NO: 114. 127, 140, 153, 166, 179, or a sequence having at least 70% identity thereto or at most 5 mutations.

In some specific embodiments, the antibody or antigen binding fragment comprises a sequence set forth in any one of groups (1) - (7):

(1) The HCDR1 comprises SEQ ID NO: 104. 107 or 109; the HCDR2 comprises SEQ ID NO: 105. 182-186, 108 or 110, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 106 or 111; and/or, the LCDR1 comprises SEQ ID NO:112 or 115, said LCDR2 comprises a sequence of any one of SEQ ID NOs 113 or 116, and said LCDR3 comprises SEQ ID NOs: 114;

(2) The HCDR1 comprises SEQ ID NO: 117. 120 or 122, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 118. 121 or 123, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 119 or 124; and/or, the LCDR1 comprises SEQ ID NO:125 or 128, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 126 or 129, and the LCDR3 comprises the sequence of any one of SEQ ID NOs: 127;

(3) The HCDR1 comprises SEQ ID NO: 130. 133 or 135, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 131. 134 or 136, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 132 or 137; and/or, the LCDR1 comprises SEQ ID NO:138 or 141, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 139 or 142, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 140;

(4) The HCDR1 comprises SEQ ID NO: 143. 146 or 148, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 144. 147 or 149, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 145 or 150; and/or, the LCDR1 comprises SEQ ID NO:151 or 154, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 152. 187 or 155, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 153;

(5) The HCDR1 comprises SEQ ID NO: 156. 159 or 161, said HCDR2 comprises the sequence of any one of SEQ ID NOs: 157. 188-189, 160 or 162, said HCDR3 comprises the sequence of any one of SEQ ID NOs: 158 or 163; and/or, the LCDR1 comprises SEQ ID NO:164 or 167, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 165 or 168, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 166;

(6) The HCDR1 comprises SEQ ID NO: 169. 172 or 174, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 170. 173 or 175, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 171 or 176; and/or, the LCDR1 comprises SEQ ID NO:177 or 180, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 178 or 181, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 179;

(7) Sequences having at least 70% identity or at most 5 mutations to each CDR in groups (1) - (6) HCDR1-3 and/or LCDR1-3;

preferably, the HCDR1-3 and/or the LCDR1-3 are determined according to Kabat, chothia or IMGT rules.

In some specific embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising the HCDR1-3 and/or a light chain variable region comprising the LCDR1-3;

preferably, the heavy chain variable region comprises: as set forth in 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 having at least 70% identity or up to 20 mutations to the sequence shown;

Preferably, the light chain variable region comprises: as set forth in 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, or a sequence having at least 70% identity or up to 20 mutations to the sequence shown;

more preferably, (1) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 15. 27, 32-40, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 16. 28-31;

more preferably, (2) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 17. 41, 46-48, and the light chain variable region comprises the sequence set forth in any one of SEQ ID NOs: 18. 42-45;

more preferably, (3) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 19. 49-51, 54-61, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 20. 52-53, 62-67;

more preferably, (4) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 21. 68, 70-72, and the light chain variable region comprises a sequence set forth in any one of SEQ ID NOs: 22. 69, 73-77;

more preferably, (5) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 23. 78, 81-87; the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 24. 79-80, 88-89;

More preferably, the heavy chain variable region of (6) comprises the amino acid sequence as set forth in SEQ ID NO: 25. 90, 93-99, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 26. 91-92, 100-103;

more preferably, (7) said heavy chain variable region and/or said light chain variable region comprises a sequence having at least 70% identity or up to 20 mutations to the sequences set forth in groups (1) - (6).

In some specific embodiments, the heavy chain variable region and/or the light chain variable region is selected from VH and/or VL set forth in table 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.

In some specific embodiments, 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; the up to 5 mutations are further preferably up to 4, 3, 2, 1 or 0 mutations; the up to 20 mutations are further preferably up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations; preferably, the mutation is an insertion, a deletion or a substitution, preferably a conservative amino acid substitution, preferably a back mutation or a hot spot mutation.

In some specific embodiments, the antibody or antigen binding fragment further comprises a heavy chain constant region and/or a light chain constant region;

preferably, the heavy chain constant region is selected from IgG, e.g., igG1, igG2, igG3 or IgG4; the IgG may be selected from human IgG, such as human IgG1 or human IgG4; the light chain constant region is selected from a kappa chain or a lambda chain, preferably a kappa chain;

more preferably, the heavy chain constant region comprises SEQ ID NO:9 or 10, and the light chain constant region comprises the sequence set forth in SEQ ID NO:12 or 13.

In some embodiments, the antibody or antigen binding fragment is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecificSpecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, fab '-SH, F (ab') ₂ Fd, fv, scFv, diabodies (diabodies) or single domain antibodies.

In some specific embodiments, the antibody or antigen binding fragment further comprises a conjugate; the conjugate may be selected from a therapeutic agent, which may be selected from a radioisotope, a chemotherapeutic agent or an immunomodulator, or a tracer, which may be selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

In some specific embodiments, the antibody or antigen binding fragment binds to a human GARP/tgfβ1 complex and/or a monkey GARP/tgfβ1 complex;

preferably, the antibody or antigen binding fragment binds to the human GARP/TGF-beta 1 complex with a KD value of less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M;

preferably, the antibody or antigen binding fragment binds to a monkey GARP/TGF-beta 1 complex with a KD value of less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M.

In some specific embodiments, the antibody or antigen binding fragment further has a binding characteristic selected from one of the following: (1) Binding to (human) GARP monomer, not to (human) tgfβ1 monomer; (2) Binding to (human) TGF-beta 1 monomer, not to (human) GARP monomer, preferably having a KD value of less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M, 1E-12M or 1E-13M binding to (human) TGF-beta 1 monomer; (3) Binding only the (human) GARP/tgfβ1 complex, not binding both the (human) GARP monomer and the (human) tgfβ1 monomer;

preferably, the binding site of the antibody or antigen binding fragment has a characteristic selected from one of the group consisting of: (1) the binding site is located in a (human) GARP monomer; (2) the binding site is located in a (human) tgfβ1 monomer; (3) The binding site is located on the (human) GARP/tgfβ1 complex and not only on GARP or tgfβ1 of said complex.

In some specific embodiments, the antibody or antigen binding fragment further has a feature selected from at least one of the following: (1) binding to a (human) GARP/tgfβ1 complex protein; (2) Binding to cells expressing the (human) GARP/tgfβ1 complex, e.g. (human) Treg cells; (3) blocking the formation of activated tgfβ1; (4) Inhibit (human) Treg cell function, e.g. inhibit SMAD2 protein phosphorylation.

In a second aspect, the present disclosure provides an antibody or antigen binding fragment that binds to GARP/tgfβ1 complex, GARP or tgfβ1, the antibody or antigen binding fragment comprising HCDR1-3, and/or LCDR1-3, the HCDR1-3 and/or the LCDR1-3 comprising a sequence shown in table 2 or table 15, respectively, or a sequence having at least 70% identity or at most 5 mutations to a sequence shown in table 2 or table 15; preferably, the HCDR1-3 and/or the LCDR1-3 are determined according to Kabat, chothia or IMGT rules.

In some specific embodiments, the antibody or antigen binding fragment binds to a GARP/tgfβ1 complex;

the HCDR1 comprises SEQ ID NO: 130. 133, 135, 143, 146, 148, 169, 172, or 174, or a sequence having at least 70% identity thereto or up to 5 mutations;

The HCDR2 comprises SEQ ID NO: 131. 134, 136, 144, 147, 149, 157, 187-188, 160, 162, 170, 173, or 175, or a sequence having at least 70% identity thereto or up to 5 mutations;

the HCDR3 comprises EQ ID NO: 132. 137, 145, 150, 158, 163, 171, or 176, or a sequence having at least 70% identity thereto or up to 5 mutations;

the LCDR1 comprises SEQ ID NO: 138. 141, 151, 154, 164, 167, 177 or 180, or a sequence having at least 70% identity thereto or up to 5 mutations;

the LCDR2 comprises a nucleotide sequence corresponding to SEQ ID NO: 139. 142, 152, 187, 155, 165, 168, 178 or 181, or a sequence having at least 70% identity thereto or at most 5 mutations;

the LCDR3 comprises SEQ ID NO: 140. 153, 179, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the antibody or antigen binding fragment comprises a sequence set forth in any one of groups (1) - (4):

(1) The HCDR1 comprises SEQ ID NO: 130. 133 or 135, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 131. 134 or 136, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 132 or 137; and/or, the LCDR1 comprises SEQ ID NO:138 or 141, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 139 or 142, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 140;

(2) The HCDR1 comprises SEQ ID NO: 143. 146 or 148, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 144. 147 or 149, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 145 or 150; and/or, the LCDR1 comprises SEQ ID NO:151 or 154, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 152. 187 or 155, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 153;

(3) The HCDR1 comprises SEQ ID NO: 169. 172 or 174, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 170. 173 or 175, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 171 or 176; and/or, the LCDR1 comprises SEQ ID NO:177 or 180, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 178 or 181, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 179;

(4) Sequences having at least 70% identity or at most 5 mutations to each CDR in groups (1) - (3) HCDR1-3 and/or LCDR 1-3.

In some specific embodiments, the antibody or antigen binding fragment specifically binds GARP;

the HCDR1 comprises SEQ ID NO: 104. 107, 109, 156, 159 or 161, or a sequence having at least 70% identity thereto or up to 5 mutations;

The HCDR2 comprises SEQ ID NO: 105. 182-186, 108, 110, 157, 188-189, 160 or 162, or a sequence having at least 70% identity thereto or at most 5 mutations;

the HCDR3 comprises EQ ID NO: 106. 111, 158 or 163, or a sequence having at least 70% identity thereto or up to 5 mutations;

the LCDR1 comprises SEQ ID NO: 112. 115, 164 or 167, or a sequence having at least 70% identity thereto or up to 5 mutations;

the LCDR2 comprises a nucleotide sequence corresponding to SEQ ID NO: 113. 116, 165 or 168, or a sequence having at least 70% identity thereto or up to 5 mutations;

the LCDR3 comprises SEQ ID NO:114 or 166, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the antibody or antigen binding fragment comprises a sequence set forth in any one of groups (1) - (3):

(2) The HCDR1 comprises SEQ ID NO: 156. 159 or 161, said HCDR2 comprises the sequence of any one of SEQ ID NOs: 157. 188-189, 160 or 162, said HCDR3 comprises the sequence of any one of SEQ ID NOs: 158 or 163; and/or, the LCDR1 comprises SEQ ID NO:164 or 167, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 165 or 168, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 166;

(3) Sequences having at least 70% identity or at most 5 mutations to each CDR in groups (1) - (2) HCDR1-3 and/or LCDR 1-3.

In some specific embodiments, the antibody or antigen binding fragment specifically binds tgfβ1, comprising a sequence set forth in any one of groups (1) - (2):

(1) The HCDR1 comprises SEQ ID NO: 117. 120 or 122, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 118. 121 or 123, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 119 or 124; and/or, the LCDR1 comprises SEQ ID NO:125 or 128, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 126 or 129, and the LCDR3 comprises the sequence of any one of SEQ ID NOs: 127;

(2) Sequences having at least 70% identity or up to 5 mutations with each CDR in group (1) HCDR1-3 and/or LCDR 1-3.

preferably, the heavy chain variable region of (1) comprises the amino acid sequence as set forth in SEQ ID NO: 15. 27, 32-40, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 16. 28-31;

preferably, (2) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 17. 41, 46-48, and the light chain variable region comprises the sequence set forth in any one of SEQ ID NOs: 18. 42-45;

preferably, (3) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 19. 49-51, 54-61, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 20. 52-53, 62-67;

preferably, the heavy chain variable region of (4) comprises the amino acid sequence as set forth in SEQ ID NO: 21. 68, 70-72, and the light chain variable region comprises a sequence set forth in any one of SEQ ID NOs: 22. 69, 73-77;

preferably, the heavy chain variable region of (5) comprises the amino acid sequence as set forth in SEQ ID NO: 23. 78, 81-87; the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 24. 79-80, 88-89;

Preferably, the heavy chain variable region of (6) comprises the amino acid sequence as set forth in SEQ ID NO: 25. 90, 93-99, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 26. 91-92, 100-103;

preferably, the heavy chain variable region and/or the light chain variable region of (7) comprises a sequence having at least 70% identity or up to 20 mutations to the sequences set forth in groups (1) - (6).

In some specific embodiments, the heavy chain variable region and/or the philic chain variable region is selected from VH and/or VL set forth in table 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.

preferably, the heavy chain constant region is selected from IgG, such as IgG1, igG2, igG3 or IgG4, which may be selected from human IgG, such as human IgG1 or human IgG4, and the light chain constant region is selected from kappa or lambda chains, preferably kappa chains;

more preferably, the heavy chain constant region comprises SEQ ID NO:9, and the light chain constant region comprises the sequence set forth in SEQ ID NO: 12.

In some embodiments of the present invention, in some embodiments, 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 (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, fab, antibody binding fragments of the whole antibody, antibody binding fragment of the whole antibody, and antibody binding fragment of the whole antibody,Fab'、Fab'-SH、F(ab') ₂ Fd, fv, scFv, diabodies (diabodies) or single domain antibodies.

In some specific embodiments, the antibody or antigen binding fragment further has a feature selected from at least one of the following: (1) binding to a (human) GARP/tgfβ1 complex protein; (2) Binding to cells expressing the (human) GARP/tgfβ1 complex, e.g. (human) Treg cells; (3) blocking the formation of activated tgfβ1; (4) Inhibit (human) Treg cell function, e.g., inhibit SMAD2 protein phosphorylation.

In a third aspect, the present disclosure provides a multispecific antigen-binding molecule comprising at least a first antigen-binding moiety comprising an antibody or antigen-binding fragment as described previously, and a second antigen-binding moiety that binds to a different target than the first antigen-binding moiety or to a different epitope of the same target; preferably, the second antigen binding moiety is an antibody or antigen binding fragment;

preferably, the additional target is 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, such as CD3 or CD16, of immune cells are recruited and/or activated.

In a fourth aspect, the present disclosure provides a Chimeric Antigen Receptor (CAR) comprising at least an extracellular antigen binding domain comprising the aforementioned antibody or antigen binding fragment or the aforementioned multispecific antigen-binding molecule, a transmembrane domain, and an intracellular signaling domain.

In a fifth aspect, the present disclosure provides an immune effector cell that expresses the foregoing chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the foregoing chimeric antigen receptor;

preferably, the immune effector cell is selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), 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;

preferably, the immune effector cell is an autoimmune effector cell or an alloimmune effector cell.

In a sixth aspect, 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.

In a fifth aspect, the present disclosure also provides a vector comprising the aforementioned nucleic acid molecule.

In a sixth aspect, the present disclosure also provides a cell comprising the aforementioned vector.

In a seventh aspect, the present disclosure also provides a method of making the aforementioned antibody or antigen-binding fragment, or multispecific antigen-binding molecule, the method comprising: (1) Culturing the aforementioned cells and/or (2) isolating antibodies or antigen-binding fragments, or multispecific antigen-binding molecules, expressed by the cells.

In an eighth aspect, the present disclosure also provides a method of preparing the aforementioned immune effector cell, the method comprising introducing a nucleic acid molecule encoding the aforementioned chimeric antigen receptor into the immune effector cell, and/or initiating expression of the chimeric antigen receptor by the immune effector cell.

In a ninth aspect, the present disclosure also provides a pharmaceutical composition comprising the aforementioned antibody or antigen binding molecule, multispecific antigen binding molecule, immune effector cell, nucleic acid molecule, vector, cell, or product made according to the aforementioned method; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.

In a tenth aspect, the present disclosure also provides the use of the aforementioned antibodies or antigen binding fragments, multispecific antigen binding molecules, immune effector cells, nucleic acid molecules, vectors, cells, pharmaceutical compositions, or products made according to the aforementioned methods, in the manufacture of a medicament for the treatment of a tgfβ -related disease; preferably, the tgfβ -related disease is selected from cancer, a fibrotic disease, inflammation, cardiovascular and cerebrovascular disease or chronic infectious disease.

In an eleventh aspect, the present disclosure also provides a method of treating a tgfβ -related disease, the method comprising administering to a subject an effective amount of a medicament comprising the aforementioned antibody or antigen binding fragment, multispecific antigen binding molecule, immune effector cell, nucleic acid molecule, vector, cell, pharmaceutical composition, or product made according to the aforementioned method, preferably the tgfβ -related disease is selected from cancer, fibrotic disease, inflammation, cardiovascular disease, or chronic infectious disease.

In a twelfth aspect, 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 made according to the aforementioned method, for use in a medicament for treating a tgfβ -related disease, preferably selected from cancer, a fibrotic disease, inflammation, cardiovascular disease, or chronic infectious disease.

Definition and description of terms

Unless defined otherwise by the present disclosure, scientific and technical terms related to the present disclosure should have meanings understood by one of ordinary skill in the art.

Furthermore, unless otherwise indicated by the present disclosure, terms in the singular of the present disclosure shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

The terms "comprising," "including," and "having" are used interchangeably herein to mean inclusion of a solution, meaning that the solution may existOther elements than those listed. It should also be understood that the use of "including," "comprising," and "having" descriptions in this disclosure also provides a "consisting of … …" approach. Illustratively, "a composition, including a and B", is to be understood as the following technical scheme: Composition consisting of A and BA kind of electronic deviceCombinations containing other components in addition to A and BThe content falls within the aforementioned "one composition".

The term "and/or" as used in this disclosure includes the meaning of "and", "or" and "all or any other combination of the elements linked by the term(s) to which it pertains.

The term "TGF-beta 1" of the present disclosure refers to transforming growth factor beta 1, which belongs to a TGF-beta 1 family member, including precursor forms (pro-TGF-beta 1), latent forms (latent TGF-beta 1), and mature forms (material TGF-beta 1). Illustratively, the tgfβ1 of the disclosure may be derived from mammals, e.g., humans, non-human primates, rodents, e.g., human tgfβ1, cynomolgus tgfβ1, and murine tgfβ1, the sequence of the tgfβ1 including, but not limited to, uniProt No.: p01137, uniprot: a0A2K5TJB2, uniprot: p04202.

The term "GARP" (glycoprotein A repetitions predominant) of the present disclosure refers to glycoprotein a-based repeats that are receptors for tgfβ1 with which the GARP/tgfβ1 complex can be formed. Illustratively, the GARPs of the present disclosure may be derived from mammals, e.g., humans, non-human primates, rodents, e.g., human GARP, cynomolgus GARP, and murine GARP, the protein sequences of which include, but are not limited to, uniProt number: q14392, uniprot number: a0A2K5X2X0, uniprot: g3XA59. In addition, the term "GARP/tgfβ1 complex", "GARP complex" or "GARP complex" in this disclosure all refer to a complex consisting of GARP and protgfβ1, or a complex consisting of GARP and latency tgfβ1.

The term "LRRC33" (Leucine-Rich Repeat-Containing Protein) in the present disclosure refers to Leucine-Rich Repeat protein 33, which belongs to the tgfβ1 receptor, with which an LRRC33/tgfβ1 complex can be formed. Illustratively, the LRRC33 of the present disclosure may be derived from a mammal, e.g., a human, a non-human primate, a rodent, e.g., human LRRC33, cynomolgus LRRC33, or murine LRRC33, the protein sequences of which include, but are not limited to, uniProt numbers: q86YC3. The term "LRRC33/tgfβ1 complex", "LRRC33complex" or "LRRC33complex" in the present disclosure all refer to a complex consisting of LRRC33 and protgfβ1 or a complex consisting of LRRC33 and latency tgfβ1.

The term "LTBP1" (Latent Transforming Growth Factor Beta Binding Protein 1) of the present disclosure refers to latent transforming growth factor beta binding protein 1, which is a tgfβ1 receptor with which LTBP1/tgfβ1 complex can be formed. Illustratively, the LTBP1 of the present disclosure may be derived from mammals, e.g., humans, non-human primates, rodents, e.g., human LTBP1, cynomolgus monkey LTBP1, and murine LTBP1, the protein sequences of which include, but are not limited to, uniProt numbers: q14766. In addition, the term "LTBP1/tgfβ1 complex", "LTBP1 complex" or "LTBP1 complex" in the present disclosure all refer to a complex consisting of LTBP1 and pro tgfβ1 or a complex consisting of LTBP1 and latency tgfβ1.

The term "specifically binds" in this disclosure refers to antigen binding molecules (e.g., antibodies) that typically specifically bind antigen and substantially the same antigen with high affinity, but do not bind unrelated antigens with high affinity. Affinity is generally reflected in equilibrium dissociation constants (equilibrium dissociation constant, KD), where a lower KD represents a higher affinity. In the case of antibodies, high affinity generally refers to having about 10 ^-7 M or less, about 10 ^-8 M or less, about 1X 10 ^-9 M or less, about 1X 10 ^-10 M or less, 1×10 ^-11 M or less or 1X 10 ^-12 KD of M or less. The KD is calculated as follows: kd=kd/Ka, where KD represents the rate of dissociation and Ka represents the rate of binding. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., biacore) or equilibrium dialysis measurements, for example, see the KD value acquisition method set forth in example 8 of the present disclosure.

The term "antigen binding molecule" is used in the broadest sense and refers to a molecule that specifically binds an antigen. Exemplary antigen binding molecules include, but are not limited to, antibodies or antibody mimics. An "antibody mimetic" refers to an organic compound or binding domain capable of specifically binding to an antigen, but not related to the structure of the antibody, and illustratively includes, but is not limited to affibody, affitin, affilin, a designed ankyrin repeat protein (DARPin), a nucleic acid aptamer, or a Kunitz-type domain peptide.

The term "antibody" is used in the broadest sense of the present disclosure to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and/or sufficient sequence from an immunoglobulin light chain variable region to be able to specifically bind to an antigen. The present disclosure "antibodies" encompasses various forms and various structures, so long as they exhibit the desired antigen binding activity. The present disclosure "antibodies" include alternative protein scaffolds or artificial scaffolds with grafted Complementarity Determining Regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced, for example, to stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., korndorfer et al 2003,Proteins:Structure,Function,and Bioinformatics,53 (1): 121-129 (2003); roque et al, biotechnol. Prog.20:639-654 (2004). 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.

The present disclosure "antibody" includes a typical "four-chain antibody" that belongs to an immunoglobulin consisting of two Heavy Chains (HC) and two Light Chains (LC); heavy chain refers to a polypeptide chain consisting 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 in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprising a heavy chain constant region CH4 domain; the light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction; the heavy chains and the light chains are connected through disulfide bonds to form a Y-shaped structure. The antigenicity of the immunoglobulin heavy chain constant region varies due to the different amino acid composition and sequence of the immunoglobulin heavy chain constant region. Accordingly, the "immunoglobulins" of the present disclosure may be divided into five classes, or isotypes of immunoglobulins, i.e., igM, igD, igG, igA and IgE, the respective heavy chains of which are the μ, δ, γ, α and epsilon chains, respectively. The same class of Ig can be divided into subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain, e.g., igG can be divided into IgG1, igG2, igG3, igG4, igA can be divided into IgA1 and IgA2. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

The present disclosure "antibodies" also includes antibodies that do not include light chains, e.g., heavy chain antibodies (HCAbs) produced by dromedarion (Camelus dromedarius), alpaca (Camelus bactrianus), alpaca (Lama glama), alpaca (Lama guanicoe), alpaca (vicuganaacos), and the like, as well as immunoglobulin neoantigen receptors (Ig new antigen receptor, igNAR) found in cartilage lines such as shark.

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 camelids, llamas, raw ostris, alpacas, sheep, rabbits, mice, rats or chondrichthyes (e.g., shark).

The disclosure of "antibodies" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., 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 antibodies obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants (e.g., containing naturally occurring mutations or produced during production of the formulation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" in this disclosure should not be construed as requiring production of the antibody or antigen binding molecule by any particular method. For example, monoclonal antibodies can be made by a variety of techniques including, but not limited to, hybridoma techniques, recombinant DNA methods, phage display techniques, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

The term "natural antibody" in this disclosure refers to an antibody that is made and paired by the immune system of a multicellular organism. The term "engineered antibody" of the present disclosure refers to an antibody obtained by genetic engineering, antibody engineering, or the like, and illustratively "engineered antibody" includes humanized antibodies, small molecule antibodies (e.g., scFv, or the like), bispecific antibodies, or the like.

The term "monospecific antibody" in this disclosure is meant to denote an antibody having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

The term "multispecific antibody" of the present disclosure refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Thus, terms such as "bispecific," "trispecific," "tetraspecific," and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.

The term "valency" in the present disclosure refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Thus, the terms "monovalent antibody", "bivalent antibody", "tetravalent antibody" and "hexavalent antibody" refer to antibodies in which one binding site, two binding sites, four binding sites and six binding sites, respectively, are present in an antibody/antigen binding molecule.

The present disclosure "full length antibody", "intact antibody" and "intact antibody" are used interchangeably throughout this disclosure to refer to a antibody having a structure substantially similar to the structure of a native antibody.

The present disclosure "antigen binding fragments" and "antibody fragments" are used interchangeably in the present disclosure, which do not possess the entire structure of an intact antibody, but only comprise local or localized variants of the intact antibody that possess the ability to bind antigen. The present disclosure of "antigen binding fragments" or "antibody fragments" includes, but is not limited to, fab '-SH, F (ab') ₂ Fd, fv, scFv, diabodies (diabodies) and single domain antibodies.

Papain digestion of whole antibodies generates two identical antigen binding fragments, called "Fab" fragments, each containing heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH 1) of the heavy chain. As such, the term "Fab fragment" of the present disclosure refers to a light chain fragment comprising the VL domain of a light chain and the constant domain (CL), and an antibody fragment of the VH domain of a heavy chain and the first constant domain (CH 1). Fab' fragments differ from Fab fragments by the addition of 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 domain carry a free thiol group. Pepsin treatment produces F (ab') with two antigen binding sites (two Fab fragments) and a portion of the Fc region ₂ Fragments.

The term "Fd" of the present disclosure refers to an antibody consisting of VH and CH1 domains. The term "Fv" in this disclosure refers to an antibody fragment consisting of single arm VL and VH domains. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

The term "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 domains are linked by a linker (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 Pluckaphun, the Pharmacology of M)onoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ Variants thereof may be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). 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.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv, forming disulfide-linked Fv (dsFv).

The term "diabody" of the present disclosure, whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between two domains of the same chain, forces the domains to pair with 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, structures 2:1121-1123 (1994)).

The terms "single domain antibody" (single domain antibody, sdAb), "VHH" and "nanobody" have the same meaning and are used interchangeably to refer to the variable region of a cloned antibody heavy chain, constructing a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed. The single domain antibody may be derived from a camelidae heavy chain antibody or a cartilage fish IgNAR.

The term "naked antibody" in this disclosure refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term "conjugated antibody" refers to an antibody conjugated to a therapeutic agent or tracer.

The term "chimeric antibody (Chimeric antibody)" in this disclosure refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular class or subclass of antibody) and another portion 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 class or subclass of antibody), but which nevertheless retains binding activity to the antigen of interest (U.S. p 4,816,567 to Cabilly et al.; morrison et al, proc.Natl. Acad. Sci. USA,81:6851 6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., human antibody).

The term "humanized antibody" of the present disclosure refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain or partially retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune responses, and the like.

The term "fully human antibody" in this disclosure refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises constant regions, the constant regions are 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 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the present disclosure "fully human antibodies" excludes antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

The term "variable region" in this disclosure refers to a region in an antibody heavy or light chain that is involved in binding the antibody to an antigen, "heavy chain variable region" is used interchangeably with "VH", "HCVR" and "light chain variable region" is used interchangeably with "VL", "LCVR". The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVR). See, e.g., 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. The term "complementarity determining region" is used interchangeably with "CDR," and generally refers to a heavy chain variable region (VH) or a light chain variable region (VL) hypervariable region (HVR) that is also referred to as a complementarity determining region because it may form a precise complementarity with an epitope in space, wherein the heavy chain variable region CDR may be abbreviated as HCDR and the light chain variable region CDR may be abbreviated as LCDR. The term "framework region" or "FR region" is interchangeable and refers to those amino acid residues in the heavy or light chain variable region of an antibody other than the CDRs. A typical antibody variable region generally consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

For further description of CDRs, reference is made to Kabat et al, J.biol.chem.,252:6609-6616 (1977); kabat et al, U.S. department of health and public service, "Sequences of proteins of immunological interest" (1991); chothia et al, J.mol.biol.196:901-917 (1987); al-Lazikani B et Al, J.mol.biol.,. 273:927-948 (1997); macCallum et al, J.mol. Biol.262:732-745 (1996); abhinannan and Martin, mol. Immunol.,45:3832-3839 (2008); lefranc M.P. et al, dev.Comp.Immunol.,27:55-77 (2003); and honeygger and Pluckthun, J.mol.biol.,309:657-670 (2001). The "CDR" of the present disclosure may be labeled and defined in a manner 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 AbRSA websites (http:// cao.labshare. Cn/AbRSA/CDRs. Php), abYsis websites (www.abysis.org/abYsis/sequence_input/key_analysis. Cgi), and IMGT websites (http:// www.imgt.org/3Dstructure-DB/cgi/Domain GapAlig. Cgi # results). CDRs of the present disclosure include overlapping (overlapping) and subsets of amino acid residues of different definition.

The term "Kabat numbering system" of the present disclosure 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).

The term "Chothia numbering system" in this disclosure generally refers to the immunoglobulin numbering system proposed by Chothia et al, which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, e.g., chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883).

The term "IMGT numbering system" of the present disclosure generally refers to a numbering system based on the international immunogenetic information system (The international ImMunoGeneTics information system (IMGT)) initiated by Lefranc et al, see Lefranc et al, dev. Comparat. Immunol.27:55-77,2003.

The term "heavy chain constant region" of the present disclosure refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding of the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, that have more conserved amino acid sequences relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. "heavy chain constant regions" include "full length heavy chain constant regions" having a structure substantially similar to that of a natural antibody constant region and "heavy chain constant region fragments" including only a portion of the "full length heavy chain constant region. Illustratively, a typical "full length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH 2 domain-CH 3 domain; when the antibody is IgE, it further comprises a CH4 domain; when an antibody is a heavy chain antibody, then it does not include a CH1 domain. Exemplary, a typical "heavy chain constant region fragment" may be selected from a CH1, fc, or CH3 domain.

The term "light chain constant region" of the present disclosure refers to the carboxy-terminal portion of an antibody light chain, which is not directly involved in binding of an antibody to an antigen, and which may be selected from a constant kappa domain or a constant lambda domain.

The term "Fc" in this disclosure refers to the carboxy-terminal portion of an antibody that is hydrolyzed by papain from an intact antibody, typically comprising the CH3 and CH2 domains of the antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from amino acid residue position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and thus the Fc region may or may not include Lys447.

The term "conserved amino acids" in this disclosure generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). Illustratively, the amino acids within each of the following groups belong to conserved amino acid residues with each other, and the substitutions of amino acid residues within a group belong to conservative amino acid substitutions:

Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (a), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K), histidine (H);

5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "identity" of the present disclosure may be calculated by: to determine the "percent identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one 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). 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 as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between two sequences varies with the same position shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) j.mol.biol.48:444-453) algorithm (available at www.gcg.com) that has been integrated into the GAP program of the GCG software package, using the Blossum62 matrix or PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1, 2, 3, 4, 5 or 6. Also for example, using the GAP program in the GCG software package (available at www.gcg.com), the percent identity between two nucleotide sequences is determined using the nws gapdna.cmp matrix and the GAP weights 40, 50, 60, 70, or 80 and the length weights 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5.

The percent identity between two amino acid sequences or nucleotide sequences can also be determined using PAM120 weighted remainder table, gap length penalty 12, gap penalty 4, using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17).

Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present disclosure may be further used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences. Such a search may be performed, for example, using the NBLAST and XBLAST programs of Altschul et al, (1990) J.mol.biol.215:403-10 (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid (SEQ ID NO: 1) molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain a gapped alignment for comparison purposes, gapped BLAST can be used as described in Altschul et al, (1997) Nucleic Acids Res.25:3389-3402. When using BLAST and empty BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The term "Chimeric Antigen Receptor (CAR)" of the present disclosure refers to an artificial cell surface receptor engineered to express and specifically bind antigen on immune effector cells, comprising at least (1) an extracellular antigen binding domain, such as a variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into immune effector cells, and (3) an intracellular signaling domain. CARs are able to redirect T cells and other immune effector cells to a selected target, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen binding domain.

The term "nucleic acid" of the present disclosure includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. In general, a nucleic acid molecule is 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 usually represented as 5 'to 3'. In the present disclosure, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. The nucleic acid molecule may be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-and double-stranded forms. Furthermore, the nucleic acid molecules described in the present disclosure may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonded or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the disclosure in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., stadler et al, nature Medicine 2017,published online 2017, 6 months 12, doi:10.1038/nm.4356 or EP 2 101 823 B1). The present disclosure "isolated" nucleic acid 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.

The term "vector" of the present disclosure 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 and that integrate into the genome of a 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".

The term "host cell" in the present disclosure refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may comprise the mutation. Included in the present disclosure are mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cells.

The term "pharmaceutical composition" of the present disclosure refers to a formulation that exists in a form that allows for the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that have unacceptable toxicity to the subject to whom the pharmaceutical composition is administered.

The term "treatment" in this disclosure refers to surgical or pharmaceutical treatment (surgical or therapeutic treatment) with the purpose of preventing, slowing (reducing) the progression of an undesired physiological change or pathology, such as cancer, in a subject. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Subjects in need of treatment include subjects already with the condition or disease and subjects prone to the condition or disease or subjects intended to prevent the condition or disease. When referring to terms slow down, alleviate, attenuate, mitigate, alleviate, etc., the meaning also includes eliminating, vanishing, non-occurrence, etc.

The term "subject" of the present disclosure refers to an organism that receives treatment for a particular disease or disorder as described in the present disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys) or non-primate mammals, that are treated for a disease or disorder.

The term "effective amount" of the present disclosure refers to an amount of a therapeutic agent that is effective to prevent or ameliorate a disease condition or progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. An "effective amount" also refers to an amount of a compound that is sufficient to alleviate symptoms, such as treating, curing, preventing or alleviating a related medical condition, or an increase in the rate of treating, curing, preventing or alleviating such conditions. When an active ingredient is administered to an individual alone, a therapeutically effective dose is referred to as the ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term "cancer" in this disclosure refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. The term "tumor" or "tumor" in this disclosure refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to in this disclosure.

The term "EC50" of the present disclosure refers to a 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 antibody at which 50% of its maximum effect is observed, and can be measured by methods known in the art.

Drawings

FIG. 1, flow cytometry analysis detects GARP expression of a stably transformed cell line hGRP-293T cell, with the ordinate showing the number of cells and the abscissa showing the fluorescence intensity;

FIG. 2, flow cytometry analysis to detect GARP expression of the stably transformed cell line hGRARP complex-293T cells, with cell number on the ordinate and fluorescence intensity on the abscissa;

FIG. 3, flow cytometry analysis to detect GARP expression of stably transformed cell lines hGRP complex-CHOK1/S cells, with cell number on the ordinate and fluorescence intensity on the abscissa;

FIG. 4, flow cytometry analysis to detect the expression of integrin αVβ6-293T cells, with cell number on the ordinate and fluorescence intensity on the abscissa;

FIG. 5 ELISA shows serum titers after immunization of mice, with OD450 on the ordinate and serum dilution on the abscissa; FIG. 5A shows SJL mouse 6 serum-free titers and FIG. 5B shows Babl/c mouse 6 serum-free titers;

FIGS. 6A-6D, ELISA for detection of binding of murine antibody Mab017 to human GARP, human GARP/TGF-beta 1 complex, human proTGF-beta 1 and human LTBP 1/TGF-beta 1 complex, respectively;

FIGS. 7A-7D, ELISA for detection of binding of murine antibody Mab087 to human GARP, human GARP/TGF-beta 1 complex, human proTGF-beta 1 and human LTBP 1/TGF-beta 1 complex, respectively;

FIGS. 8A-8D, ELISA for detection of binding of murine antibody Mab138 to human GARP, human GARP/TGF-beta 1 complex, human proTGF-beta 1 and human LTBP 1/TGF-beta 1 complex, respectively;

FIGS. 9A-9D, ELISA for detection of binding of murine antibody Rab171 to human GARP, human GARP/TGF-beta 1 complex, human proTGF-beta 1 and human LTBP 1/TGF-beta 1 complex, respectively;

FIGS. 10A-10B, ELISA for detection of chimeric antibody ch-Mab195 binding to human GARP, human GARP/TGF-beta 1 complex, respectively;

FIG. 11 ELISA detection of chimeric antibody ch-Mab201 binding to human GARP/TGF-beta 1 complex;

FIG. 12 ELISA assay for humanized antibody binding to human GARP-his protein;

FIG. 13 ELISA assays for binding capacity of humanized antibodies to human proTGF-beta 1;

FIG. 14 ELISA assays for the binding capacity of humanized antibodies to human GARP/TGF-beta 1 complex;

FIG. 15 ELISA assays for the binding capacity of humanized antibodies to human LTBP 1/TGF-beta 1 complex;

FIG. 16 ELISA assays for the binding capacity of humanized antibody H087-H2L2 to human LRRC 33/TGF-beta 1 complex;

FIG. 17 ELISA assays for humanized antibodies binding to cynomolgus GARP/TGF-beta 1 complex;

FIG. 18 FACS detects binding of humanized antibodies to cynomolgus GARP/TGF-beta 1 complex at cellular level;

FIGS. 19A-19B, FACS detect binding of humanized antibodies to human GARP-293T cells or human GARP-complex-293T cells;

FIG. 20, flow cytometry analysis detects binding of humanized antibodies to endogenous cell L428;

fig. 21A-21B, flow cytometry analysis detects binding of humanized antibodies to human Treg cells;

FIG. 22, luciferase reporter system detects inhibition of TGF- β1 secretion by antibodies;

FIGS. 23A-23D, percentage of Treg cells pSMAD 2;

fig. 24A-24B, anti-hGARP/tgfβ1 antibodies inhibited human Treg function in vivo, wherein fig. 24A is a GVHD score result and fig. 24B is a survival result;

FIG. 25, wild type C57 mouse pharmacokinetics;

figure 26, human GARP transgenic mouse pharmacokinetics.

Detailed Description

The present disclosure is further described below in conjunction with specific embodiments, and advantages and features of the present disclosure will become apparent as the description proceeds. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The disclosed embodiments are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It will be understood by those skilled in the art that various changes and substitutions may be made in the details and form of the technical solutions of the present disclosure without departing from the spirit and scope of the present disclosure, but these changes and substitutions fall within the scope of the present disclosure.

Unless otherwise specified, each of H017-H2dL2, GARP-H017-H2dL2 and GARP-hu017-H2dL2 shown in the following examples and the accompanying drawings represents the same humanized antibody, each of H087-H2L2, GARP-H087-H2L2 and GARP-hu087-H2L2 represents the same humanized antibody, each of H138-H8L5, GARP-hu138-H8L5 represents the same humanized antibody, each of H171-H3L5, GARP-H171-H3L5 and GARP-hu171-H3L5 represents the same humanized antibody, each of H195-H3L1, GARP-hu195-H3L1 represents the same humanized antibody, and each of H201-H5L3, GARP-H201-H5L3 and GARP-H201-H201-L5 represents the same humanized antibody.

Example 1 preparation of recombinant proteins and antibodies and purification methods thereof

1.1 design and expression of recombinant proteins

Construction of GARP and protgfβ1 recombinant proteins from human, cynomolgus and mouse amino acid sequences: the human GARP protein (UniProt number: Q14392) and human TGF beta 1 precursor protein (UniProt number: P01137) are taken as template sequences, fusion proteins with labels are designed, cloned to pTT5 vectors respectively, GARP plasmids and proTGF beta 1 plasmids are constructed, and transiently expressed in HEK293 cells to obtain antigens and proteins for detection of the present disclosure. Separately transfecting GARP and proTGF beta 1 plasmids into HEK293 cells to obtain corresponding monomer proteins; the GARP/TGF beta 1 complex can be obtained by co-transfection after mixing GARP and proTGF beta 1 plasmids. The preparation method of the cynomolgus monkey and mouse proteins is the same as the preparation method of the human recombinant proteins. Cynomolgus monkey GARP sequence is from Uniprot number: the TGF-beta 1 precursor protein sequence is from Uniprot: a0A2K5TJB2. Mouse GARP sequences are from Uniprot: the G3XA59, TGF-beta 1 precursor protein sequence is from Uniprot: p04202. Specific sequence information for GARP and progfβ1 recombinant proteins are shown below:

human GARP-his protein (his tagged human GARP protein extracellular domain fusion protein) (SEQ ID NO: 1):

Human proTGF-beta 1-his protein (his tagged human TGF-beta 1 precursor fusion protein) (SEQ ID NO: 2):

cynomolgus monkey GARP-Flag/his (Flag/his tagged cynomolgus monkey GARP extracellular domain fusion protein) (SEQ ID NO: 3):

cynomolgus monkey proTGF-beta 1 (cynomolgus monkey TGF-beta 1 precursor protein sequence without tag) (SEQ ID NO: 4):

mouse mGRARP-Flag/His (Flag/His tagged mouse GARP protein extracellular region) (SEQ ID NO: 5):

His-mTGF beta 1-pro (His-tagged mouse TGF beta 1 precursor protein) (SEQ ID NO: 6):

recombinant proteins constructed from the human LRRC33 and LTBP1 amino acid sequences: human LRRC33 protein (UniProt: Q86YC 3) and human LTBP1 protein (UniProt: Q14766) are used as template sequences, fusion proteins with his labels are designed and cloned into pTT5 vectors respectively, then plasmids of LRRC33 and LTBP1 are respectively transfected into HEK293 cells together with the proTGF beta 1 plasmids, after seven days of culture, cell supernatants are collected and purified, and then human LRRC33/TGF beta 1 complexes and human LTBP1/TGF beta 1 complexes (also called human LTBP1 complexes) are obtained. Specific sequence information of the recombinant protein is shown as follows:

hLRRC33-ECD-his (his tagged human LRRC protein extracellular domain fusion protein) (SEQ ID NO: 7):

hLTBP1-ECD-his (human LTBP1 protein extracellular domain fusion protein with his tag) (SEQ ID NO: 8):

1.2 design and expression purification of antibodies

The control antibodies employed in the present disclosure are derived from published patent sequences. ARGX-115, SRK-Ab6, SRK181 and 28G11 control antibodies were expressed recombinantly using human IgG 4+kappa subtype, unless otherwise indicated. The h151D control antibody was expressed recombinantly using human igg1+κ subtype. As an exception, the control antibodies used in the identification of the murine antibodies described in example 3 were all recombinantly expressed using the mouse migg2a+κ subtype (fig. 6-9). The chimeric and humanized antibodies of murine mab described in example 3 of the present disclosure were all recombinantly expressed using the human igg4+κ 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 to an expression vector pTT5, HEK293 cells are transiently transfected, cell supernatants are collected for Protein A antibody purification after shaking culture for 7 days at 37 ℃, and the purification process is described in "1.3.2 Protein A affinity chromatography purification hybridoma supernatant/chimeric antibody/humanized antibody". Specific sources and sequence information of antibodies are shown in Table 1.

Table 1 control antibody sequence listing

1.3 purification of recombinant proteins, hybridoma antibodies, and purification of recombinant antibodies

1.3.1 Nickel column purification of recombinant proteins

After constructing and expressing the relevant recombinant protein according to the step "design and expression of recombinant protein 1.1", purification was performed as follows: and centrifuging the cell expression supernatant sample at a high speed to remove impurities. The nickel column was equilibrated with 20mM PBS+500mM NaCl solution and washed 2-5 column volumes. The supernatant samples are combined on columns, and nickel columns of different companies can be selected as the medium. The column was washed with equilibration solution until the A280 reading reduced to baseline, and then eluted with equilibration solutions containing 10mM,20mM,40mM,90mM,250mM,500mM imidazole, respectively, and the individual elution peaks were collected and the fractions of the protein of interest were determined from SDS-PAGE gel. The collected elution product containing the target protein can be further purified by gel chromatography Superdex200 (GE), the mobile phase is PBS, the peak of the polymer and the impurity protein is removed, and the elution peak of the target product is collected. The obtained protein is split into separate parts for standby after electrophoresis and peptide drawing, LC-MS identification are right. Proteins purified by this protocol include human GARP-His, human proTGF beta 1-His, human GARP/TGF beta 1 complex, cynomolgus GARP/TGF beta 1 complex, human LRRC33/TGF beta 1 complex, and human LTBP1/TGF beta 1 complex.

1.3.2 Protein A affinity chromatography purification of hybridoma supernatants/chimeric/humanized antibodies

The cell culture supernatant expressing the antibody was first harvested by high-speed centrifugation. The Protein A affinity column was washed 3-5 column volumes with 0.1M NaOH and then 3-5 column volumes with pure water. The column was equilibrated 3-5 column volumes using a 1 XPBS (pH 7.4) buffer system as an equilibration buffer. The cell supernatants were loaded for binding at low flow rates, the flow rates were controlled to allow retention times of about 1min or longer, and after binding, the column was washed 3-5 column volumes with 1 XPBS (pH 7.4) until UV absorbance fell back to baseline. Sample elution was performed using 50mM citric acid/sodium citrate (pH 3.0-3.5) buffer, elution peaks were collected according to UV detection, and the eluted product was rapidly adjusted to pH 5-6 using 1M Tris-HCl (pH 8.0) for buffer storage. For the eluted product, solution displacement may be performed by methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution displacement to a desired buffer system, or desalting using a molecular exclusion column such as G-25 to a desired buffer system, or removing the polymer component in the eluted product using a high resolution molecular exclusion column such as Superdex 200 to increase the sample purity.

Example 2 construction of stably transfected cell lines

The nucleotide sequence encoding the full-length amino acid sequence of human GARP (UniProt: Q14392) was cloned into the pcDNA3.1-hygromycin vector (available from Clontech) and plasmids were prepared and designated as encoding the full-length plasmid of human GARP. The nucleotide sequence encoding human full-length proTGF-beta 1 (UniProt: P01137) was cloned into pcDNA3.1-puromycin vector (available from Clontech) and plasmids were prepared and designated as encoding human TGF-beta 1 full-length plasmids.

Transfection of HEK-293T cell line (purchased from ATCC) with full-length plasmid encoding human GARP3000 Transfection Kit, available from Invitrogen, cat: l3000-015) was selectively cultured in DMEM medium containing 0.5mg/ml Hygromycin and 10% (w/w) fetal bovine serum for 2 weeks, using PE anti-human GARP antibodies (BioLegend, cat: 352504 Positive monoclonal cells were sorted on a flow cytometer FACSAriaIII (available from BD Biosciences) to 96-well plates and placed at 37 ℃,5% (v/v) CO ₂ After about 2 weeks of incubation, a portion of the monoclonal wells was selected for amplification. Clones after amplification were screened by flow cytometry. The monoclonal cell line with better growth vigor and higher fluorescence intensity is selected to be further amplified and cultured and stored in liquid nitrogen for later use, and the obtained cell line is named hGRARP-293T (figure 1).

The full-length plasmid encoding human GARP and full-length plasmid encoding human TGF-beta 1 were co-transfected into HEK293T cells, and after pressurized screening of hygromycin and puromycin, monoclonal selection, FACS identification with ARGX-115 antibody, the resulting cell line was designated hGARP complex-293T (FIG. 2). The same procedure was used to stably transform CHOK1/S cells and the resulting cell line was designated hGRP-complex CHOK1/S. FACS detection cell line expression is shown in FIG. 3.

The full-length amino acid sequence encoding human integrin subunit ITGAV (Uniprot: P06756) was cloned into the pcDNA3.1-hygromycin vector (available from Clontech) and plasmids were prepared. The full-length amino acid sequence encoding human integrin subunit ITGB6 (Uniprot: P18564) was cloned into pcDNA3.1-Neo vector and plasmids were prepared. The ITGAV and ITGB6 plasmids are adopted3000 After simultaneous transient transformation of HEK293T cells by the Transfection Kit (available from Invitrogen, cat# L3000-015), in DMEM medium containing 0.5mg/ml Hygromycin and G418, 10% (w/w) fetal bovine serumSelective incubation for 2 weeks, positive monoclonal cells were sorted on a flow cytometer FACSariaIII (from BD Biosciences) with PE anti-human integrin αVβ6 antibody (Abcam, cat# ab 77906) to 96-well plates and placed at 37 ℃,5% (V/V) CO ₂ After about 2 weeks of incubation, the monoclonal wells were selected for amplification. Clones after amplification were screened by flow cytometry. And selecting a monoclonal cell line with better growth vigor and higher fluorescence intensity, continuing to perform expansion culture and performing liquid nitrogen freezing storage for standby, wherein the obtained cell line is named as integrin alpha V beta 6-293T. FACS detection cell line expression is shown in FIG. 4.

Example 3 preparation of antibodies

3.1 immunization of animals

Monoclonal antibodies of the present disclosure are produced by immunizing mice. The experimental mice were 6-8 week old, female SPF grade Balb/C mice or SJL mice (purchased from Charles River). The immunogen is human GARP/TGF-beta 1 complex protein or hGRP-complex CHOK1/S stably transfected cell line prepared in example 1. At the time of primary immunization of proteomes, the immunogens were emulsified with TiterMax (ex Sigma, T2684-1M) and injected Subcutaneously (SC) and Intraperitoneally (IP) with 0.1mL each, i.e., 50 μg immunogen per mouse; at boost, the immunogen was injected subcutaneously and intraperitoneally with ImjectAlum (ex Thermo) in 0.1ml, i.e. 25 μg of immunogen per mouse. During primary immunization of a cell group, titerMax is emulsified with physiological saline and then injected into the abdominal cavity with 0.1mL, and after 15min, 0.1mL of cell suspension is injected into the abdominal cavity, namely each mouse is injected with 1X 10 ⁷ A cell; when the immunity is enhanced, the cell quantity of the intraperitoneal injection is 1 multiplied by 10 ⁷ Is described. Immunization was performed weekly, and blood was taken on days 3, 19, 47, 61. Binding to human GARP/tgfβ1 complex protein (His tag, coating concentration 4 μg/mL) was detected by ELISA method, and the presence and antibody titer of antibodies recognizing human GARP/tgfβ1 complex in serum of immunized animals were tested. According to the serum titer, mice with high antibody titer in serum and a plateau-like titer were selected for spleen cell fusion. Serum assay titers are shown in FIGS. 5A-5B. The immunization was boosted 3 days before the spleen cell fusion, and 50. Mu.g/saline-formulated antigen solution was subcutaneously and intraperitoneally injected.

Monoclonal antibodies against human GARP/tgfβ1 can also be generated by immunizing rats, experimental rats 6-8 weeks old SD rats, purchased from Shanghai Laike. The immunogen adopts human GARP/TGF beta 1 complex protein, the dosage of the immunogen is doubled compared with the dosage of mice, and 100 mug is first avoided. The immunization method and the fusion screening method are identical to those of mice. Rab171 antibodies in the present disclosure were obtained by immunization of rats.

3.2 cell fusion

Spleen and lymph nodes were aseptically removed, ground and filtered with a 40 μm cell strainer (from BD Falcon), ACK Lysing Buffer (from Gibco) was added, the spleen cells were lysed to spiked erythrocytes, a spleen cell suspension was obtained, and cells were washed 1 time by centrifugation at 1500rpm with DMEM (from Gibco) basal medium. Then, the mixture was mixed with mouse myeloma cell SP2/0 (purchased from ATCC) at a ratio of 2:1 in terms of the number of living cells, washed 2 times with Cytofusion Medium C (purchased from BTX), and subjected to cell fusion by the BTX ECM2001+ electrofusion method (see ECMU 2001+ ELECTROFUSION PROTOCOL). The fused cells were diluted into DMEM medium containing 20% fetal bovine serum (purchased from ExCell Bio), 1 xhat (purchased from Sigma). Spleen cells were then cultured at 2X 10 ⁴ Each well was added to 96-well cell culture plates and incubated at 37℃with 5% CO ₂ In an incubator. After 10 days, the supernatant of the cell fusion plate was screened by ELISA, and positive clones detected by ELISA were amplified to 24-well plates for expansion culture. After 3 days of culture, the culture broth from the 24-well plate was used to determine the binding activity to human GARP/TGF-beta 1 complex protein by ELISA and FACS.

Based on the 24-well plate screening results, eligible hybridoma cells were selected for subcloning in 6-well plates using Medium D (available from stemcel). Monoclonal was picked 7 days after subcloning in DMEM medium with 10% fbs, 1×ht (purchased from Sigma) at 37 ℃,5% co ₂ The cells were cultured for 2 days, and positive monoclonal amplifications were selected to 24-well plates for further culture, and after 2 days, antigen binding positive selection criteria were determined by ELISA and FACS. Based on the 24-well plate sample detection results, the optimal clone was selected and isolated in DMEM medium containing 10% FBS at 37℃in 5% CO ₂ And (3) performing expansion culture on the optimal clone under the condition, and performing liquid nitrogen freezing storage to obtain the hybridoma cell.

3.3 hybridoma antibody screening

After 7-10 days of fusion, taking hybridoma cell supernatant, detecting the combination of the hybridoma cell supernatant and human GARP/TGF beta 1 complex protein by a protein ELISA method, and selecting positive clones; the following day positive clones were tested for binding to hGRP-293T cells, human GARP-his protein, human proTGF beta 1 protein, human GARP complex-293T cells by protein and cell ELISA. Clones with higher positive signals of the primary screening are selected for subcloning, and the hybridoma antibodies after subcloning are detected by the same method. Positive clones were classified according to binding, 3 classes total: (1) Clones that bind only to the GARP/tgfβ1 complex and do not bind to GARP or progfβ1; (2) Clones that bind to the GARP/tgfβ1 complex and protgfβ1, but do not bind to GARP; (3) Clones that bind to the GARP/tgfβ1 complex and GARP monomers but do not bind to protgfβ1. Finally, 201 hybridoma cells are obtained through screening and identification, and all the 201 antibodies can recognize the human GARP/TGF beta 1 complex.

3.4 production of murine monoclonal antibodies

From the 201 hybridoma clones after the strain fixing, 6 hybridomas with the strongest binding capacity were selected for expansion culture (Mab 017, mab087, mab138, rab171, mab195, mab 201), and the supernatant was collected and centrifuged and the antibody was purified as described in example 1.3.2.

Example 4 hybridoma antibody sequencing

Cloning of antibody gene sequences from positive hybridoma cells was as follows: collecting hybridoma cells in logarithmic growth phase, extracting RNA with Trizol (Invitrogen, cat No. 15596-018) according to the procedure of kit instruction, and extracting RNA with PrimeScript ^TM Reverse Transcriptase kit 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 sequenced by sequencing company. The murine antibodies Mab017, mab087, mab138, rab171, mab195, mab201 were sequenced and the Heavy Chain Variable Region (HCVR) and Light Chain Variable Region (LCVR) amino acid sequences are shown below.

Mab017 HCVR(SEQ ID NO:15)：

Mab017 LCVR(SEQ ID NO：16)：

Mab087 HCVR(SEQ ID NO:17)：

Mab087 LCVR(SEQ ID NO:18)：

Mab138 HCVR(SEQ ID NO：19)：

Mab138 LCVR(SEQ ID NO：20)：

Rab171 HCVR(SEQ ID NO：21)：

Rab171 LCVR(SEQ ID NO：22)：

Mab195 HCVR(SEQ ID NO：23)：

Mab195 LCVR(SEQ ID NO：24)：

Mab201 HCVR(SEQ ID NO：25)：

Mab201 LCVR(SEQ ID NO：26)：

Analysis of the CDR regions of the antibodies by bioinformatics, in particular byKabat numbering system and Chothia numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)AndIMGT numbering System (http:// www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. Cgi) The specific results are shown in Table 2.

TABLE 2 murine antibody CDR sequences and numbering

Example 5 construction of human IgG4 chimeric antibody

According to the sequencing results of the hybridoma antibodies Mab017, mab087, mab138, rab171, mab195, mab201 and the variable region coding genes thereof obtained in examples 3-4, primers were designed, and the VH/VL gene fragments of each antibody were constructed by PCR using the sequencing genes as templates. The obtained VH/VL gene fragment is subjected to homologous recombination with an expression vector pTT5 (with a signal peptide and a human IgG4 constant region/kappa constant region gene (CH 1-Fc/CL) fragment), and a recombinant chimeric antibody full-length expression plasmid pTT5-VH-CH1-Fc/pTT5-VL-CL is constructed to form six chimeric antibodies of CH-Mab017, CH-Mab087, CH-Mab138, CH-Rab171, CH-Mab195 and CH-Mab 201. Wherein the amino acid sequences of the human IgG4 constant and kappa constant regions are set forth in Table 1. Antibody heavy and light chains were as per 1 after plasmid preparation: 1 into Expi293F cells, shaking at 37℃for 7 days, collecting the supernatant, centrifuging and purifying the antibody according to the purification method described in example 1.3.2.

Example 6 humanized design of murine antibody

6.1 antibody humanized variable region design

By comparing IMGT (http:// IMGT. Cines. FR) human antibody heavy and light chain variable region germline gene database and MOE (Molecular Operating Environment ) software, heavy and light chain variable region germline genes with high homology with murine antibody are respectively selected as templates, and CDRs (CDRs determined by Kabat numbering system (http:// www.abysis.org/analysis/sequence_input/key_analysis. Cgi)) of the murine antibody are respectively transplanted into corresponding human templates to form the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Variable region sequences. Back mutation and/or hot spot mutation are performed as needed.The antibody sequences of this example were carried out according to the Kabat numbering system Sequence numbering and determination of CDR regions thereof. Unless otherwise indicated, the antibody sequences of this example are shown in italics for framework regions, bold + underlined for CDR regions, bold + bold border for back mutations or hot spot mutations.

1. Humanization of Mab017

1.1, mab017 framework selection

Humanized light chain templates of the murine antibody Mab017 are IGKV1-39 x 01 and IGKJ2 x 01, humanized heavy chain templates are IGHV1-3 x 01 and IGHJ6 x 01, and CDRs of the murine antibody Mab017 are respectively transplanted into the humanized templates, so that the humanized antibody h017 of the Mab017 is obtained, and the variable region sequences of the humanized antibody h017 are shown as follows.

h017 HCVR(VH-CDR graft，IGHV1-3*01)：

h017 LCVR(VL-CDR graft，IGKV1-39*01)：

1.2 Back-mutation and Hot-spot mutation designs of Mab017 humanized antibodies

According to the requirement, key amino acids in the FR region sequence of the mAb017 humanized antibody are subjected to back mutation to ensure the original affinity, and meanwhile, in view of the fact that a high-risk easy-modification site NNGG exists in the heavy chain CDR2 region of the mAb017, the NG is subjected to amino acid mutation to eliminate the molecular modification risk, and the specific mutation design is shown in Table 3.

TABLE 3 humanized antibody back-mutation and Hot-spot mutation designs of Mab017

And (3) injection: graft represents implantation of murine antibody CDRs into human germline FR region sequences; graft+A43S means that position A of Graft is mutated to S, and so on.

1.3, mab017 humanized antibodies

The humanized antibody back mutations and hot spot mutation designs of Table 3 Mab017 were combined to finally obtain various humanized antibodies of Mab017 (see Table 4 for details).

TABLE 4 Mab017 humanized antibody VH-VL pairing modes

	h017L1	h017L2	h017L3	h017L4
h017H1	h017H1L1	h017H1L2	h017H1L3	h017H1L4
h017H2	h017H2L1	h017H2L2	h017H2L3	h017H2L4
h017H2a	N/A	h017H2aL2	N/A	N/A
h017H2b	N/A	h017H2bL2	N/A	N/A
h017H2c	N/A	h017H2cL2	N/A	N/A
h017H2d	N/A	h017H2dL2	N/A	N/A
h017H2e	N/A	h017H2eL2	N/A	N/A
h017H3	h017H3L1	h017H3L2	h017H3L3	h017H3L4
h017H4	h017H4L1	h017H4L2	h017H4L3	h017H4L4

Note that: H017H1L1 means that 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.

The amino acid sequences of H017L1, H017L2, H017L3, H017L4, H017H1, H017H2a, H017H2b, H017H2c, H017H2d, H017H2e, H017H3 and H017H4 are as follows.

h017L1 (same as h017 LCVR) (SEQ ID NO: 28):

h017L2(SEQ ID NO：29)：

h017L3(SEQ ID NO：30)：

h017L4(SEQ ID NO：31)：

h017H1(SEQ ID NO：32)：

h017H2(SEQ ID NO：33)：

h017H2a(SEQ ID NO：34)：

h017H2b(SEQ ID NO：35)：

h017H2c(SEQ ID NO：36)：

h017H2d(SEQ ID NO：37)：

h017H2e(SEQ ID NO：38)：

h017H3(SEQ ID NO：39)：

h017H4(SEQ ID NO：40)：

2. humanization of Mab087

2.1, mab087 framework selection

Humanized light chain templates of the murine antibody Mab087 are IGKV4-1 x 01, IGKV2D-29 x 01 and IGKJ4 x 01, humanized heavy chain templates are IGHV2-26 x 01 and IGHJ6 x 01, and CDRs of the murine antibody Mab087 are respectively transplanted into the humanized templates, so that the humanized antibody h087 of the Mab087 is obtained. The h087 variable region sequence is shown below.

h087 HCVR(VH-CDR graft,IGHV2-26*01)(SEQ ID NO：41)：

h087 LCVR1(VL-CDR graft 1,IGKV4-1*01)(SEQ ID NO：42)：

h087 LCVR2(VL-CDR graft 2,IGKV2D-29*01)(SEQ ID NO：43)：

2.2 reverse mutation design of humanized antibodies to Mab087

According to the requirement, key amino acids in the FR region sequence of the humanized antibody of the mAb087 are subjected to back mutation to amino acids corresponding to the murine antibody so as to ensure the original affinity, and the specific back mutation design is shown in Table 5.

TABLE 5 humanized antibody back mutation design for Mab087

Note that: graft represents implantation of murine antibody CDRs into human germline FR region sequences; a93G means that the shift position 93 a of Graft is mutated to G, and so on.

2.3, mab087 humanized antibodies

The above-described Mab087 humanized antibody back-mutation designs of table 5 were combined to finally obtain various Mab087 humanized antibodies, see table 6 for specific combinations.

TABLE 6 Mab087 humanized antibody VH-VL pairing modes

Note that: H087H1L1 means that 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.

Specific sequences of H087L1, H087L2, H087L3, H087L4, H087H1, H087H2 and H087H3 are shown below.

h087L1(SEQ ID NO：42)：

h087L2(SEQ ID NO：44)：

h087L3(SEQ ID NO：43)：

h087L4(SEQ ID NO：45)：

h087H1(SEQ ID NO：46)：

h087H2(SEQ ID NO：47)：

h087H3(SEQ ID NO：48)：

3. Humanization of Mab138

3.1 Mab138 framework selection

Humanized light chain templates of the murine antibody Mab138 are IGKV1-39 x 01, IGKV4-1 x 01 and IGKJ2 x 01, humanized heavy chain templates are IGHV3-7 x 01, IGHV3-30 x 02, IGHV3-15 x 01 and IGHJ6 x 01, and CDRs of the murine antibody Mab138 are respectively transplanted into the humanized templates, so that humanized antibody H138 of the Mab138 is obtained, and the variable region sequence is as follows.

Hab138 HCVR1(VH-CDR graft1，IGHV3-7*01)(SEQ ID NO：49)：

Hab138 HCVR2(VH-CDR graft2，IGHV3-30*02)(SEQ ID NO：50)：

Hab138 HCVR3(VH-CDR graft3，IGHV3-15*01)(SEQ ID NO：51)：

H138 LCVR1(VL-CDR graft1，IGKV1-39*01)(SEQ ID NO：52)：

H138 LCVR2(VL-CDR graft2，IGKV4-1*01)(SEQ ID NO：53)：

3.2 Reverse mutation design of humanized antibody of Mab138

According to the requirement, key amino acids in the FR region sequence of the humanized antibody of the Mab138 are subjected to back mutation into amino acids corresponding to the murine antibody so as to ensure the original affinity, and the specific back mutation design is shown in Table 7.

TABLE 7 humanized antibody variable region back mutation design of Mab138

Note that: graft represents implantation of murine antibody CDRs into human germline FR region sequences; L46A denotes mutation of L at position 46 of Graft to A, and so on.

3.3 Mab138 humanized antibody:

the humanized antibody back-mutation designs of Mab138 of table 7 above were combined to finally obtain a variety of Mab138 humanized antibodies, see table 8 for specific combinations.

TABLE 8 Mab138 humanized antibody VH-VL pairing modes

	h138H1	h138H2	h138H3	h138H4	h138H5	h138H6	h138H7	h138H8
h138L1	h138H1L1	h138H2L1	h138H3L1	h138H4L1	h138H5L1	N/A	N/A	N/A
h138L2	h138H1L2	h138H2L2	h138H3L2	h138H4L2	h138H5L2	N/A	N/A	N/A
h138L3	h138H1L3	h138H2L3	h138H3L3	h138H4L3	h138H5L3	h138H6L3	h138H7L3	h138H8L3
h138L4	N/A	N/A	h138H3L4	N/A	N/A	h138H6L4	h138H7L4	h138H8L4
h138L5	N/A	N/A	h138H3L5	N/A	N/A	h138H6L5	h138H7L5	h138H8L5
h138L6	N/A	N/A	h138H3L6	N/A	N/A	h138H6L6	h138H7L6	h138H8L6

Note that: H138H1L1 means that 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.

Specific sequences of H138H1, H138H2, H138H3, H138H4, H138H5, H138H6, H138H7, H138H8, H138L1, H138L2, H138L3, H138L4, H138L5, H138L6 are shown below.

h138H1(SEQ ID NO：54)：

h138H2(SEQ ID NO：55)：

h138H3(SEQ ID NO：56)：

h138H4(SEQ ID NO：57):

h138H5(SEQ ID NO：58):

h138H6(SEQ ID NO：59):

h138H7(SEQ ID NO：60):

h138H8(SEQ ID NO：61):

h138L1(SEQ ID NO：62)：

h138L2(SEQ ID NO：63):

h138L3(SEQ ID NO：64):

h138L4(SEQ ID NO：65):

h138L5(SEQ ID NO：66):

h138L6(SEQ ID NO：67):

4. Humanization of Rab171

4.1 Rab171 framework selection

Humanized light chain templates of the murine antibody Rab171 are IGKV1-39 x 01 and IGKJ2 x 01, humanized heavy chain templates are IGHV3-30 x 01 and IGHJ3 x 01, and CDRs of the murine antibody Rab171 are respectively transplanted into the humanized templates, so that the humanized antibody h171 of Rab171 is obtained, and the variable region sequences of the humanized antibody h171 are 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)：

4.2, reverse mutation and hot spot mutation design of humanized antibody of Rab 171:

according to the requirement, key amino acids in the FR region sequence of the humanized antibody of Rab171 are subjected to back mutation to amino acids corresponding to the murine antibody so as to ensure the original affinity; meanwhile, a high-risk easy-modification site NG exists in the light chain CDR2 region based on Rab171, so that the NG is subjected to amino acid mutation according to the antibody structure in a computer simulation mode to eliminate the molecular modification risk, and the specific mutation design is shown in Table 9.

Table 9 humanized antibody back-mutation and hot spot mutation designs of Rab171

Note that: graft represents implantation of murine antibody CDRs into human germline FR region sequences; A43S denotes mutation of Graft position 43A to S, and so on.

4.3, rab171 humanized antibody:

the above-described Rab171 humanized antibody back mutation and hot spot mutation designs of table 9 were combined to finally obtain various Rab171 humanized antibodies (see table 10 for details).

TABLE 10 Rab171 humanized antibody VH-VL pairing modes

	h171H1	h171H2	h171H3
h171L1	h171H1L1	h171H2L1	h171H3L1
h171L2	h171H1L2	h171H2L2	h171H3L2
h171L3	h171H1L3	h171H2L3	h171H3L3
h171L4	h171H1L4	h171H2L4	h171H3L4
h171L5	h171H1L5	h171H2L5	h171H3L5

Note that: H171H1L1 denotes that Rab171 humanized antibody H171 has a light chain variable region as described for H171L1 and a heavy chain variable region as described for H171H1, and so on.

Specific sequences of H171H1, H171H2, H171H3, H171L1, H171L2, H171L3, H171L4, and H171L5 are shown below.

h171H1(SEQ ID NO：70)：

h171H2(SEQ ID NO：71)：

h171H3(SEQ ID NO：72):

h171L1(SEQ ID NO：73)：

h171L2(SEQ ID NO：74)：

h171L3(SEQ ID NO：75):

h171L4(SEQ ID NO：76):

h171L5(SEQ ID NO：77):

5. Humanization of Mab195

5.1, mab195 framework selection

Humanized light chain templates of the murine antibody Mab195 are IGKV1-NL1 x 01, IGKV2-28 x 01 and IGKJ4 x 01, humanized heavy chain templates are IGHV1-18 x 01 and IGHJ6 x 01, and CDRs of the murine antibody Mab195 are respectively transplanted into the humanized templates, so that the humanized antibody h195 of the Mab195 is obtained, and the variable region sequence of the humanized antibody h195 is as follows.

h195 HCVR (also known as VH-CDR shift, IGHV1-18 x 01) (SEQ ID NO: 78):

h195 LCVR1 (also known as VL-CDR graft1, IGKV1-NL1 x 01) (SEQ ID NO: 79):

h195 LCVR2 (also known as VL-CDR graft2, IGKV2-28 x 01) (SEQ ID NO: 80):

5.2, reverse mutation and hot spot mutation design of humanized antibody of Mab 195:

according to the requirement, key amino acids in the FR region sequence of the humanized antibody of the Mab195 are subjected to back mutation to amino acids corresponding to the murine antibody so as to ensure the original affinity; meanwhile, because the high-risk easy-modification site NG exists in the mAb195 heavy chain CDR2 region, the NG is subjected to amino acid mutation according to the antibody structure in a computer simulation mode to eliminate the molecular modification risk, and the specific mutation design is shown in Table 11.

Table 11 humanized antibody back-mutation and hot spot mutation designs of Mab195

5.3, mab195 humanized antibody:

the humanized antibody back mutation and hot spot mutation designs of the Mab195 of the above table 11 were combined to finally obtain various humanized antibodies of Mab195, as detailed in table 12.

TABLE 12 Mab195 humanized antibody VH-VL pairing modes

	h195H1	h195H2	h195H3	h195H4	h195H5	h195H6	h195H7
h195L1	h195H1L1	h195H2L1	h195H3L1	h195H4L1	h195H5L1	h195H6L1	h195H7L1
h195L2	h195H1L2	h195H2L2	h195H3L2	h195H4L2	h195H5L2	h195H6L2	h195H7L2

Note that: H195H1L1 means that Mab195 humanized antibody H195 has a light chain variable region as described for H195L1 and a heavy chain variable region as described for H195H1, and so on.

The amino acid sequences of H195H1, H195H2, H195H3, H195H4, H195H5, H195H6, H195H7, H195L1, H195L2 are shown below.

h195H1(SEQ ID NO：81)：

h195H2(SEQ ID NO：82)：

h195H3(SEQ ID NO：83)：

h195H4(SEQ ID NO：84)：

h195H5(SEQ ID NO：85)：

h195H6(SEQ ID NO：86)：

h195H7(SEQ ID NO：87)：

h195L1(SEQ ID NO：88)：

h195L2(SEQ ID NO：89)：

6. Humanization of Mab201

6.1, mab201 framework selection

Humanized light chain templates of the murine antibody Mab201 are IGKV3-11 x 01, IGKV6-21 x 01 and IGKJ2 x 01, humanized heavy chain templates are IGHV1-8 x 01 and IGHJ6 x 01, and CDRs of the murine antibody Mab201 are respectively transplanted into the humanized templates to obtain the humanized antibody h201 of the Mab201, wherein the variable region sequences are as follows.

h201 HCVR(VH-CDR graft，IGHV1-8*01)(SEQ ID NO：90)：

h201 LCVR1(VL-CDR graft1，IGKV3-11*01)(SEQ ID NO：91)：

h201 LCVR2(VL-CDR graft2，IGKV6-21*01)(SEQ ID NO：92)：

6.2, reverse mutation design of humanized antibody of Mab 201:

according to the requirement, key amino acids in the FR region sequence of the humanized antibody of the Mab201 are subjected to back mutation into amino acids corresponding to the murine antibody so as to ensure the original affinity, and the specific back mutation design is shown in Table 13.

Humanized antibody back mutation design of Table 13 Mab201

Note that: graft represents implantation of murine antibody CDRs into human germline FR region sequences; L48W denotes mutation of L at position 48 of Graft to W, and so on.

6.3, mab201 humanized antibody:

the humanized antibody back mutation designs of the mabs 201 of the above table 13 were combined to finally obtain various humanized antibodies of the mabs 201, as shown in table 14.

TABLE 14 Mab201 humanized antibody VH-VL pairing modes

	h201H1	h201H2	h201H3	h201H4	h201H5	h201H6	h201H7
h201L1	h201H1L1	h201H2L1	h201H3L1	N/A	N/A	N/A	N/A
h201L2	h201H1L2	h201H2L2	h201H3L2	N/A	N/A	N/A	N/A
h201L3	h201H1L3	h201H2L3	h201H3L3	h201H4L3	h201H5L3	h201H6L3	h201H7L3
h201L4	h201H1L4	h201H2L4	h201H3L4	N/A	N/A	N/A	N/A
h201L5	h201H1L5	h201H2L5	h201H3L5	h201H4L5	h201H5L5	h201H6L5	h201H7L5

Note that: H201H1L1 represents that 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.

The amino acid sequences of H201H1, H201H2, H201H3, H201H4, H201H5, H201H6, H201H7, H201L1, H201L2, H201L3, H201L4, and H201L5 are as follows.

h201H1(SEQ ID NO：93)：

h201H2(SEQ ID NO：94)：

h201H3(SEQ ID NO：95)：

h201H4(SEQ ID NO：96)：

h201H5(SEQ ID NO：97)：

h201H6(SEQ ID NO：98)：

h201H7(SEQ ID NO：99)：

h201L1(SEQ ID NO：100)：

h201L2(SEQ ID NO：101)：

h201L3(SEQ ID NO：102)：

h201L4(SEQ ID NO：103)：

h201L5(SEQ ID NO：91)：

7. The CDR sequences (determined according to the Kabat numbering system) of Mab017, mab087, mab138, mab 171, mab195 and Mab201 humanized antibodies are summarized in the following table:

TABLE 15 humanized antibody CDR sequences and numbering thereof

EXAMPLE 7 preparation of humanized full Length antibody

7.1 plasmid construction of humanized full Length antibody

The gene fragment encoding each humanized antibody VH/VL of example 6 was subjected to homologous recombination with the expression vector pTT5-huIgG4HC or pTT5-huIgGLC (Kappa) (with signal peptide and constant region gene) by gene synthesis, to construct an antibody full-length expression vector. The heavy and light chain constant region amino acid sequences are shown in table 1.

7.2 expression purification of humanized full Length antibodies

The constructed heavy chain and light chain plasmids of the antibody are respectively prepared according to the following steps of 1:1 into an Expi293F cell, shaking the cell with 120rpm in an incubator at 37℃for 7 days, collecting the cell supernatant, centrifuging the cell supernatant and purifying the antibody by using protein A according to the step 1.3.1.

Example 8 affinity assay for antibodies

8.1 murine antibody affinity assay

The binding strength of the antibodies to the antigen was detected by anti-murine antibody capture using a BIAcore 8K instrument. First, the Anti-Mouse IgG antibody was immobilized on a CM5 chip using an amino coupling method according to the direction of Mouse Antibody Capture Kit kit, the chip was activated for about 600 seconds after mixing NHS and EDC using HBS-EP+pH7.4 as a mobile phase, the Anti-Mouse IgG antibody was diluted to 15. Mu.g/mL with 10mM sodium acetate pH5.0, injected for 420 seconds, and finally the remaining activation sites were blocked with ethanolamine. Then, the affinity of the antibodies to the antigen was determined using multicycle kinetics, in each cycle, the antibody to be tested was first captured with anti-murine antibody, then a single concentration of human GARP/tgfβ1 protein complex antigen (prepared from example 1) was injected, the binding and dissociation processes of the antibody and antigen protein were recorded, and finally chip regeneration was completed with Glycine ph1.7, wherein the mobile phase was HBS-ep+ ph7.4 (10mM HEPES,150mM NaCl,3mM EDTA,0.05%surfactant P20), flow rate was 30 μl/min, regeneration time was 30 seconds, detection temperature was 25 ℃. Finally, the data are analyzed according to a 1:1 binding model, and the equilibrium dissociation constant KD is obtained by fitting the antigen binding kinetic parameters of the antibody. See table 16 for specific test results.

Table 16 affinity of murine antibodies to hGRP/TGF-beta 1 complexes

Antibody name	KD
Mab017	7.59E-11M
Mab087	3.18E-10M
Mab138	1.95E-10M
Rab171	1.29E-09M
Mab195	1.21E-10M
Mab201	4.60E-10M
ARGX-115-mIgG2a	2.90E-09M

8.2 chimeric and humanized antibody affinity assays

The binding strength of the antibodies to the antigen was detected using a BIAcore 8K instrument using the Protein A capture method. First, protein A was immobilized on CM4 chip by amino coupling method, after mixing NHS and EDC using HBS-EP+pH7.4 as mobile phase according to the direction of Amine Coupling Kit kit, the chip was activated for about 600 seconds, protein A was diluted to 50. Mu.g/mL with 10mM sodium acetate pH4.5, injected for 600 seconds, and finally the remaining activation sites were blocked with ethanolamine. Then, the affinity of the antibody to the antigen was determined by multi-cycle kinetics, in each cycle, the chimeric antibody to be tested (example 5) or the humanized antibody was first captured by a Protein A chip (example 7), then a single concentration of human GARP/TGF-beta 1 Protein complex or cynomolgus GARP/TGF-beta 1 Protein complex or human proTGF-beta 1 monomeric antigen (prepared in example 1) was injected, the binding and dissociation processes of the antibody and antigen Protein were recorded, and finally chip regeneration was completed by Glycine pH1.5, wherein the mobile phase was HBS-EP+ (10mM HEPES,150mM NaCl,3mM EDTA,0.05%surfactant P20), the flow rate was 30. Mu.L/min, the regeneration time was 30s, and the detection temperature was 25 ℃; finally, the data were analyzed according to the 1:1binding model, fitting the antibody antigen binding kinetic parameters, obtaining the equilibrium dissociation constant KD.

Affinity with human GARP/tgfβ1 protein: 017 humanized antibody combinations (Table 4) all had affinities less than 7.11E-09M;087 humanized antibody combinations (Table 6) all have less than 3.38E-11M affinity; 138 humanized combinations (Table 8) all had affinities less than 9.07E-09M; the 171 humanized antibody combination (Table 10) had an affinity of less than 8.07E-09M; the 201 humanized antibody combinations (Table 14) all have less than 1.66E-09M affinity; the 195 humanized antibody combination (Table 12) all had an affinity of less than 5.69E-10M.

Humanized combinations that maintain chimeric antibody affinities or affinity decreases within a factor of 2 were selected as final molecules and tested for affinity to cynomolgus GARP/tgfβ1 protein complex or to human protgfβ1 monomers (see table 17 for details).

Table 17 affinity assay results for chimeric and humanized antibodies

Antibody numbering	KD (human GARP/TGF beta 1)	KD (cynomolgus monkey GARP/TGFbeta 1)	KD (human proTGF beta 1 monomer)
h017-H2dL2	1.24E-10M	1.53E-10M	/
ch-Mab017	2.23E-10M	2.85E-10M	/
h138-H8L5	2.76E-09M	4.41-09M	/
ch-Mab138	2.17E-09M	3.94-09M	/
h171-H3L5	3.00E-10M	1.29E-08M	/
ch-Mab171	1.71E-10M	5.32E-09M	/
h195-H3L1	1.41E-10M	2.66E-10M	/
ch-Mab195	1.07E-10M	2.44E-10M	/
h201-H5L3	1.31E-09M	1.791E-09M	/
ch-Mab201	1.37E-09M	1.97E-09M	/
Control antibody ARGX-115	2.05E-09M	3.80E-09	/
h087-H2L2	8.94E-12M	2.53E-11M	1.62E-13M
ch-Mab087	6.86E-12M	2.25E-11M	NotTest
Control antibody SRK-Ab6	4.25E-10M	7.44E-10M	9.48E-11M

Note that: the "/" in table 17 indicates "not bonded".

Example 9 ELISA detection of antibody binding to human GARP protein, proTGF-beta 1 protein, GARP/TGF-beta 1 protein Complex, LTBP 1/TGF-beta 1 protein Complex, LRRC 33/TGF-beta 1 protein Complex

The 6 antibody molecules of the present disclosure have different binding properties to GARP proteins, protgfβ1 proteins, and GARP/tgfβ1 complexes. The ELISA detection method used in the present disclosure is as follows: diluting antigen protein (prepared in example 1) to 4. Mu.g/mL with PBS pH7.4, adding ELISA plate (burning, CAT# 9018), 50. Mu.L/well, overnight at 4 ℃, throwing away coating solution, adding 5% skimmed milk powder (Bio, CAT#A 600669-0250) -PBS, 250. Mu.L/well, incubating at 37℃for 2-4 hours, washing with 0.05% Tween20-PBS (bio, CAT#A100777-0500, B548117-0500) on a plate washer (Biotek, CAT#A100777-0500, B548117-0500), adding dilutions of purified antibodies (murine purified antibodies, human-murine chimeric antibodies, humanized antibodies were all diluted to 2. Mu.g/mL with 1% BSA, 12 concentration points diluted with 3-fold continuous gradient), incubating at 37℃for 1.5-2 hours, HRP enzyme-labeled antibody (rat murine sample antibody was Jackson, CAT#112-035-003, mouse murine sample antibody was Jackson, CAT#115-035-003, human-mouse chimeric antibody, humanized antibody was Merck, CAT#AP113P) was washed three times with 0.05% Tween20-PBS at a dilution ratio of 1:5000, TMB color-developing solution (KPL, CAT#52-00-03), 50. Mu.L/well, room temperature for 10 minutes, 1M HCL, 50. Mu.L/well, termination reaction, and OD450nm was read by an enzyme-labeling instrument (PE, enht or EnVision) by adding to the ELISA plate, incubating at 37℃for 1 hour, washing with 0.05% Tween20-PBS three times on the plate. And (3) injection: and eliminating the data value of the abnormal hole in the data processing process.

By this method, the binding of the murine antibody Mab017 to human GARP, proTGF-beta 1, GARP/TGF-beta 1 complex and LTBP 1/TGF-beta 1 complex was detected, respectively, as shown in FIGS. 6A to 6D. Mab017 hardly binds to the GARP-his protein and the proTGF-beta 1 protein, but binds well to the GARP/TGF-beta 1 complex. However, as shown in example 12, humanized 017 antibodies could bind to GARP-293T cells at the cellular level (FIG. 19A), presumably due to the conformational differences of the GARP-His protein and the cell membrane protein. The binding properties of such antibodies are similar to the control antibody h151D, i.e., the binding site of the antibody is on the GARP monomer.

The binding of murine antibody Mab087 to human GARP, proTGFβ1, GARP/TGFβ1 complex, LTBP1/TGFβ1 complex is shown in FIGS. 7A-7D. Mab087 can bind to human proTGF-beta 1 monomers and complexes formed by proTGF-beta 1 (human GARP/TGF-beta 1 complex, human LTBP 1/TGF-beta 1 complex) without binding to GARP-His monomers. Such antibodies have similar binding properties to the control antibody 28G11, i.e. the binding site is on the protgfβ1 monomer.

The binding of murine antibody Mab138 to human GARP, proTGFβ1, GARP/TGFβ1 complex, LTBP1/TGFβ1 complex is shown in FIGS. 8A-8D. Mab138 binds neither GARP-his nor protgfβ1 protein, only to the complex formed by GARP/tgfβ1, i.e. the binding site is on both GARP and protgfβ1, and such antibody binding properties are similar to the control antibody ARGX-115.

The binding of murine antibody Rab171 to human GARP, proTGFβ1, GARP/TGFβ1 complex, LTBP1/TGFβ1 complex is shown in FIGS. 9A-9D. Rab171 did not bind to the GARP monomer nor to the proTGF-beta 1 monomer, only to the complex formed by GARP/TGF-beta 1, and the binding characteristics of the antibody were similar to that of Mab138 and the control antibody ARGX-115, with binding sites on both GARP and proTGF-beta 1.

During the hybridoma supernatant phase, mab195 is known to bind to GARP and GARP/tgfβ1 complex, but not progfβ1 (specific quantitative data not shown). The binding of chimeric antibody ch-Mab195 to human GARP and GARP/TGF-beta 1 complex is shown in FIGS. 10A-10B, the chimeric antibody ch-Mab195 can partially bind to GARP protein, has good binding activity to GARP/TGF-beta 1 complex, and the binding site is on GARP monomer.

The hybridoma supernatant screening stage is known to have no binding of Mab201 to both GARP monomer and protgfβ1, only to GARP/tgfβ1 complex (specific quantitative data not shown), binding sites on both GARP and protgfβ1. ELISA detects the binding of chimeric antibody ch-Mab201 to the human GARP/TGF-beta 1 complex, as shown in FIG. 11. The binding capacity of the chimeric antibody ch-Mab201 to the human GARP/TGF-beta 1 complex was comparable to that of the control antibody ARGX-115.

The binding capacity of the engineered humanized antibodies to human GARP, protgfβ1, GARP/tgfβ1 complex, LTBP1/tgfβ1 and LRRC33/tgfβ1 complex was further tested by ELISA. The detection method of the humanized antibody is consistent with that of the murine antibody, and the use of the secondary antibody is changed from anti-mouse to anti-human.

As shown in FIG. 12, the binding capacities of the humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1, H201-H5L3 and the control antibodies to human GARP-his proteins were examined. The H017-H2dL2 and H195-H3L1 antibodies may bind to the GARP-his protein, while the other antibodies do not bind to the GARP-his protein.

As shown in FIG. 13, the binding capacities of the humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1, H201-H5L3 and the control antibodies to human proTGF beta 1-his proteins were examined. H087-H2L2 can be well combined with proTGF beta 1 protein.

As shown in FIG. 14, the binding capacities of the humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1, H201-H5L3 and the control antibodies to the human GARP/TGF-beta 1 complex proteins were examined. The humanized antibodies described above are all capable of binding efficiently to the GARP/tgfβ1 complex.

As shown in FIG. 15, the binding capacities of the humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1, H201-H5L3 and the control antibodies to the human LTBP 1/TGF-beta 1 complex proteins were examined. ELISA detection results show that the H087-H2L2 and LTBP1/TGF beta 1 complex have good binding capacity.

As shown in FIG. 16, ELISA detects the binding capacity of the humanized antibody H087-H2L2 to the LRRC 33/TGF-beta 1 complex. H087-H2L2 can well bind to the complex formed by LRRC33/TGF beta 1.

Example 10 ELISA detection of binding of antibodies to cynomolgus monkey GARP/TGF beta 1 Complex

The humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1, H201-H5L3 and the control antibodies were analyzed for binding to the cynomolgus GARP/TGF-beta 1 protein complex (prepared from example 1) according to the ELISA detection method described in example 9. As shown in FIG. 17 and Table 18, the aforementioned antibodies had excellent cross-binding activity to the cynomolgus GARP/TGF-beta 1 complex.

Table 18 results of ELISA assays for binding of antibodies to cynomolgus GARP/TGF-beta 1 complexes

Antibody name	Emax	EC50,nM
ARGX-115-hIgG4	2.377	0.07441
GARP-h138-H8L5	2.22	0.1801
GARP-h201-H5L3	1.929	0.572
GARP-h171-H3L5	2.111	0.06003
GARP-h017-H2dL2	2.45	0.0466
GARP-h195-H3L1	2.358	0.04423
GARP-h087-H2L2	2.628	0.03747
SRK-Ab6-hIgG4	2.405	0.09779
28G11-hIgG4	2.351	0.09868
Anti-Hel-hIgG4	0.086	N/A

Note that: N/A indicates poor fit or invalid fit

Example 11 FACS detection of binding Activity of antibodies to cells expressing cynomolgus monkey GARP/TGF-beta 1 Complex

293T cells were collected, 5X 10 ⁶ The individual cells were seeded into 15cm cell culture dishes (burning, CAT # 430599), 5% CO at 37 ℃ ₂ After overnight incubation, fresh 10% FBS-DMEM medium (excel, CAT#FSP500; gibco, CAT#11995) was changed the next day, and cynomolgus GARP plasmid and cynomolgus proTGFbeta 1 plasmid were transiently expressed according to the liposome 3000 transfection kit (invitrogen, L3000-015) instructions, and after 48 hours of transient, cells were collected, washed once with PBS (Hyclone, CAT#SH 30256), and 1% BSA-PBS was resuspended to 2X 10 ⁵ mu.L, 1% BSA-PBS diluted antibody to 270nM, 3-fold serial gradient diluted 12 concentration points, 50. Mu.L cells and 50. Mu.LMixing L antibody dilutions, incubating at 4deg.C for 60 min, washing twice with PBS, adding Alexa647 fluorescein-labeled secondary antibody (1:800) (humanized antibody using Jackson, CAT # 109-605-088), 100. Mu.L/well resuspended cells, incubated at 4℃for 40 min, washed twice with PBS, resuspended with 1% BSA-PBS, 100. Mu.L/well and cell samples analyzed on a flow cytometer (BD, canto Plus or Canto II). As shown in FIG. 18 and Table 19, H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1, H201-H5L3 bound cynomolgus GAPR/TGF-beta 1 complex well at the cellular level.

Table 19 results of FACS detection of binding of antibodies to cells expressing cynomolgus GARP/TGF-beta 1 Complex

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

Note that: N/A represents the difference or lack of fit

EXAMPLE 12 FACS detection of antibody binding to human GARP-293T cells overexpressing GARPcomplex-293T cells

The over-expression cell line constructed in example 2 was used. Cells were collected, washed once with PBS (Hyclone, CAT#SH 30256), and resuspended to 2X 10 in 1% BSA-PBS ⁵ mu.L, 1% BSA-PBS diluted antibody to 270nM, 3-fold serial gradient dilution of 12 concentration points, 50. Mu.L cells were mixed with 50. Mu.L antibody dilution, incubated at 4℃for 60 min, PBS washed twice, Alexa addition647 fluorescein labeled secondary antibody (1:800) (rat murine sample antibody with Jackson, CAT #112-605-003, mouse murine sample antibody with Jackson, CAT #115-605-003, human-murine chimeric antibody, humanized antibody with Jackson, CAT # 109-605-088), 100. Mu.L/well resuspended cells, incubated at 4℃for 40 min, PBS washed twice, resuspended with 1% BSA-PBS, 100. Mu.L/well, and cell samples analyzed on a flow cytometer (BD, canto Plus or Canto II). The results of the detection are shown in FIGS. 19A to 19B and tables 20 to 21.

The 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 FIG. 19A and Table 20. Wherein the H017-H2dL2 and H195-H3L1 antibodies bind GARP monomers well at the cellular level.

Table 20 results of FACS detection of binding of antibodies to human GARP-293T cells

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

Note that: N/A represents no fit or a difference in fit

The binding of humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H201-H5L3, H195-H3L1 to human GARP complex-293T overexpressing cells is shown in FIG. 19B and Table 21, and all antibodies tested bound well to the GARP/TGF beta 1 complex.

Table 21 results of FACS detection of binding of antibodies to GARP Complex-293T cells

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

Note that: N/A represents no fit or a difference in fit

EXAMPLE 13 FACS detection of antibody binding Activity to endogenous cell L428

Using the FACS detection method of example 11, L428 cells were collected, the binding activity to L428 cells was detected by adding the corresponding antibodies, and flow analysis was performed to generate a fluorescence intensity profile. As shown in FIG. 20, humanized antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H201-H5L3, H195-H3L1 bind well to endogenously expressed GARP/TGF beta 1 complex.

EXAMPLE 14 flow cytometry analysis of antibody binding to human Treg cells

Human Treg cells were isolated from human PBMC using a sorting kit (Stemcell, cat# 18063), expanded for 15 days by in vitro stimulation with Dynabeads Human Treg Expander (Gibco, cat# 11129D) and frozen in sub-packs. The Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300×g for 5min the next day, resuspended in DPBS to give a cell suspension, and counted. The number of cells required for the experiment was added to a centrifuge tube, centrifuged at 300 Xg for 5min, the supernatant was removed, and the cell density was adjusted to 2X 10 with staining buffer (2% FBS in PBS v/v)) ⁶ Per ml, 50 μl per well of 96 well plate was plated. All test antibodies and control antibodies anti-Hel isotype (diluted with staining buffer to a maximum concentration of 10. Mu.g, respectively) were taken Per ml, then 5-fold dilution, total 8 concentrations). Test and control antibodies were added to each well, 50 μl per well. The well plate with the cell suspension and antibody added is placed on a microplate shaker and shaken at 500rpm for 1min, and the cells and antibody are thoroughly mixed. After mixing well, the well plate was placed in a refrigerator at 4 ℃ and incubated for 30min. After the incubation, 100. Mu.l of staining buffer was added to each well, centrifuged at 350 Xg for 5min, and the supernatant was discarded; cells were resuspended by adding 200. Mu.l staining buffer per well, centrifuged at 350 Xg for 5min, and the supernatant discarded. Adding PE coat anti-Human IgG Fc (eBioscience, cat# 12-4998-82) into a staining buffer solution according to the proportion of 250:1 (staining buffer solution: corresponding staining fluorescent antibody) to prepare a staining solution, uniformly mixing, and adding into cell holes with 100 mu l per hole; the well plate was placed on a microplate shaker and shaken at 500rpm for 1min to thoroughly mix the cells with the dye solution. Mixing, placing in a refrigerator at 4deg.C, and incubating for 30min. Cells were washed twice and finally resuspended in 150 μl PBS per well and the signal was detected on-stream (Thermo Attune NxT). Simultaneously detecting expression of GARP and LAP on the surfaces of Treg cells: 0.8million cells were resuspended in 50. Mu.l DPBS, 50. Mu.l 2 Xlive/dead violet (Invitrogen, cat. L34964; final concentration 1:500 dilution) was added and stained at 4℃for 20min. The staining buffer was washed once. Mu.l of diluted PE anti-hGRP (bioleged, cat# 352504; 2. Mu.l/test) and APC anti-hLAP (bioleged, cat# 349106; 2.5. Mu.l/test) were added, the Isotype control antibodies PE-mIgG2b, kappa Isotype (bioleged, cat# 400314) and APC-mIgG1, kappa Isotype (BD, cat# 554681) were stained at 4℃for 30min, and the cells were washed once with staining buffer. The cells were resuspended in 150. Mu.l and the signal was detected on-stream (Thermo Attune NxT). As shown in FIGS. 21A-21B, H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1 and H201-H5L3 were all able to bind effectively to Treg cells. The binding fluorescence intensity of the antibodies to Treg cells is shown in table 22.

Table 22 antibodies bind to human Treg cells

Note that: "/" means "not fit or fit difference"

EXAMPLE 15 anti-GARP/TGF-beta 1 Complex antibodies blocking secretion of activated TGF-beta 1 by GARP complex-293T cells

The experimental procedure employed in this disclosure is as follows: the first day, purified antibody samples were diluted 3-fold with medium (MEM, gibco,11095, +0.5% fbs,1% non-essential amino acids, gibco,11140,1mm sodium pyruvate, 1% penicillin, gibco,15140,) at 9 concentration points from 50 nM; collecting hGRP-complex-293T stably transformed cells and alpha vβ6-293T stably transformed strain cells, and re-suspending the culture medium; antibody and resuspended cells were added sequentially to 96-well cell culture plates (corning, CAT # 3799), 5% CO at 37 ℃C ₂ Culturing for 24 hours. The next day of the experiment, TGF beta/SMAD signaling pathway SBE reporter cells-HEK 293 (TGF beta/SMAD Signaling Pathway SBE Reporter-HEK293 Cell Line, BPS, CAT#60653) were collected, resuspended in 96-well Cell culture plates (corning, CAT#3599), 100. Mu.L/well, 5% CO at 37℃with recovery medium (MEM, gibco,11095, +10% FBS+1% nonessential amino acids, gibco,11140, +1mM nonessential amino acids, gibco,11140, +1% penicillin, gibco, 15140) ₂ Culturing for 24 hours.

Discarding culture solution of SBE report cell-HEK 293 of TGF beta/SMAD signal path, transferring antibody sample, GARP complex-293T cell and alpha v beta 6-293T co-incubation culture solution into report gene cell culture plate, 100 μl/well, 37 ℃ 5% CO ₂ Culturing. Then, bright-gro luciferase assay system (promega, cat#e 2620), 100 μl/well, and blow to allow sufficient lysis of reporter cells, then transfer to 96-well flat-bottom white-light-emitting plate (corning, cat#3610), 150 μl/well, and read luminescence signal with enzyme-labeled instrument (PE, enVision).

In this experiment, the magnitude of the fluorescein signal may represent the secreted amount of active tgfβ1. From the results, it can be seen (FIG. 22, table 23) that antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H201-H5L3, H195-H3L1 in the present disclosure all significantly inhibited secretion of activated TGF-beta 1.

Table 23 antibodies inhibit secretion of activated TGF-beta 1

Antibodies to	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

Note that: N/A represents "no fit or fit difference"

EXAMPLE 16 anti-human GARP/TGF-beta 1 antibodies TGF-beta 1 factor inhibiting human Treg cell production Activity

Treg cells secrete tgfβ1 factor, which upon binding to the receptor activates downstream signaling, leading to phosphorylation of SMAD2 protein. p-SMAD2, upon phosphorylation, forms a complex with pSMAD3 and SMAD4, and nuclear entry regulates transcription of a range of downstream genes. The level of p-SMAD2 phosphorylation may represent the degree of activation of signaling pathways, reflecting the amount of active tgfβ1 factor produced by human Treg cells.

Human Treg cells were isolated from human PBMC using a sorting kit (Stemcell, cat# 18063), expanded for 15 days by in vitro stimulation with Dynabeads Human Treg Expander (Gibco, cat# 11129D) and frozen in sub-packs. The in vitro isolated expanded Treg cells were recovered overnight, centrifuged at 300×g for 5 min the next day, resuspended in AIM-V medium (Gibco, cat No. 31035025) to give a cell suspension, and counted. The number of cells required for the experiment was taken and the cell density was adjusted to 2X 10 ⁶ Per ml, 96-well plates were plated, 50. Mu.l per well, and then anti-Hel isotype (diluted to 60. Mu.g/ml with AIM-V, respectively) was added at different concentrations of the test antibody and the negative control antibody, with a final concentration of 10. Mu.g/ml. Dynabeads Human Treg Expander (Gibco, cat# 11129D) was washed once with AIM-V and the concentration was adjusted to 1X 10 ⁶ Per ml, 50. Mu.l of the 96-well plate was added to each well. Meanwhile, positive control wells were set, 20. Mu.l/well hTGF-. Beta.1 (Peprotech, cat. No.: 100-21) was added to the positive control wells (diluted to 60ng/ml with AIM-V, final concentration 10 ng/ml), and Treg only wells, i.e., treg cells without drug and Dynabeads, were set as negative controls. The cells and the antibody are fully and evenly mixed by lightly blowing by a multichannel pipetting gun, and after being evenly mixed, the pore plate is placed in 5 percent CO ₂ The incubator was incubated at 37℃for 48 hours. After the end of the incubation, the well plate was removed and the cells and Dynabeads were blown off with a multichannel pipette, centrifuged at 350 Xg for 4 minutes, and the supernatant was discarded. Mu.l DPBS was added to resuspend cells per well, 50. Mu.l 2 Xlive/dead violet (Invitrogen, cat. L34964, final 1:500 dilution) was added per well, and stained at 4℃for 20min. With Incubation buffer (0.5 g BSA+1)00ml PBS) was washed twice. Centrifuge 350g,4min, discard supernatant. Add 100. Mu.l/well BD Phosflow preheated at 37 ℃ ^TM Fix Buffer I (BD, cat# 557870) was fixed at 37℃for 10min. Cells were washed twice with Incubation buffer, centrifugation speed increased to 400g,4min and supernatant discarded. Then BD Phosflow was added slowly at 100. Mu.l/Kong Yuleng ^TM Perm Buffer III (BD, cat# 558050) resuspended cells and reacted on ice for 30min. Cells were washed twice, 100. Mu.l/well of anti-pSMAD2 antibody diluted with incubation buffer (CST, cat# 26945S, 2. Mu.l/test) was added, and a PE-Rabbit IgG Isotype control (CST, cat# 5742S) of equivalent mass concentration was prepared. The reaction was kept away from light at room temperature for 1 hour. Cells were washed twice, 150 μ l incubation buffer resuspended in each well and the signal was detected on stream (Thermo Attune NxT). After homogenization, SMAD2 phosphorylation is shown in fig. 23A-23D.

As shown in fig. 23A-23D, antibodies H017-H2dL2, H087-H2L2, H138-H8L5, H171-H3L5, H195-H3L1 and H201-H5L3 of the present disclosure were effective in inhibiting phosphorylation of SMAD2 by Treg cells, indicating that they were effective in inhibiting tgfβ1 factor, which is an activity of human Treg cell production.

Example 17 anti-GARP/TGF-beta 1 antibodies inhibit human Treg cell function in vivo

To assess whether the antibodies could inhibit Treg in vivo, human PBMCs were transferred to severely immunodeficient mice (NPG: female 5-6 weeks, beijing vergta biotechnology limited)), and xenogeneic GVHD models were established. In this model, tregs are important immune cells that regulate the rate and extent of GHVD occurrence, which if downregulated would promote GVHD occurrence.

PBMC cells were purchased from the eriosema chinense organism and autologous tregs were expanded using the following method: treg cells were isolated from PBMC of the same donor using Stem cell human CD4+CD127lowCD25+ Treg isolation kit (cat# 18063) and then expanded in vitro using Dynabeads (bead-to-cell ratio at 2:1) and hIL-2 (200 IU/mL) and 1. Mu.M Rapamycin for 7 days without the addition of Rapamycin after 7 days, treg cells were collected after 16 days of expansion and stained with HuCD45, CD3, CD4, CD25, foxP3 antibodies conjugated with fluorochromes. Treg cell purity (purity > 93%) was determined by flow cytometry and expanded Treg cells were frozen for later use.

At-1 day, 10 animals per group were randomly grouped and administered 1 time per week, 6 times per week, and 20mg/kg. Then on day 0, tail vein expanded human tregs (5×10 per mouse ⁶ ) Mixed human PBMC (5X 10 per mouse) ⁶ ). On day 14 and day 28, each of the re-tail vein injections of expanded human tregs (2.5×10 per mouse ⁶ ). GVHD development in mice was measured 1 week for the first two weeks of dosing and observation period, and GVHD development in mice was measured 2 weeks for the last 4 weeks. The GVHD score was established based on: weight loss (0 point:<10%, 1:: 10% -20%, 2::>20%, 3:>30%), anemia (0 score: red or pink tails; 1, the method comprises the following steps: white tail), pose (0 points: normally, 1 point: humpback), general activity (0 points: normally, 1 point: limited), dehairing (0 points: no depilation, 1:: dehairing) and jaundice (0 score: white or red tail, 1 point: yellow tail). The maximum disease severity or death corresponds to 7 points. In this example and the accompanying drawings, H138, H017 and H087 refer to humanized full length antibodies H138-H8L5, H017-H2dL2 and H087-H2L2, respectively, prepared according to example 7.

See fig. 24A-24B and table 24 for specific results. As shown in fig. 24A, by day 36 after the first dose, the GVHD score curve was higher overall in mice dosed with h017 than in PBS group, starting from the onset of GVHD (day 18) significantly earlier than in PBS control group (day 29), while the GVHD score (5.63 points) was significantly higher in h017 group at day 36 than in PBS control group (1.88 points).

As shown in table 24, mice given h087 had significantly higher GVHD scores than PBS control (p < 0.001) based on statistical analysis of GVHD. GVHD scores were higher in mice in the h138 dosing group than in the control PBS group (p < 0.01).

Fig. 24A shows that the GVHD score of mice in the h 017-dosed group was significantly higher than that of the ARGX-115-dosed group compared to the control antibody ARGX-115 (×p < 0.01); the GVHD score of mice in the h 087-dosed group was significantly higher than that of ARGX-115-dosed group (p < 0.001).

The occurrence of GVHD resulted in a shortened survival time of the host mice, as shown in fig. 24B survival analysis: the overall survival of mice was consistent with the trend of GVHD scores. Compared with the PBS of the control group, the survival rate of mice with the administration of h087, h017 and h138 is obviously reduced, which indicates that the antibodies of h087, h017 and h138 inhibit the immune cell function of human Treg in the mice, thereby promoting GVHD to occur.

Table 24 GVHD score statistics

Example 18 pharmacokinetics of antibodies in mice

18.1 pharmacokinetic evaluation in Normal mice

To compare the pharmacokinetic differences between the different antibodies, SPF-grade female wild-type C57 mice were used in this example, 6-8 weeks old, weighing about 18-20g, 3 in each group, were given a single tail intravenous dose of 10mg/kg of control antibody (ARGX-115, SRK-Ab 6) and 6 of the humanized antibodies prepared by example 7.2 (H138-H8L 5, H017-H2dL2, H087-H2L2, H171-H3L5, H201-H5L3, H195-H3L 1).

The mice are fed with standard feed without fasting and water-stopping. The medicine is diluted by normal saline. The eyebox was sampled at the time points of 0.25 hours, 2 hours, 24 hours, 72 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours before, after, and after, administration. After blood samples were collected in micropipettes and allowed to stand for about 30min, the samples were centrifuged at 12000rpm at 4℃for 5 min, serum was separated into low adsorption centrifuge tubes, compound codes and time points were indicated and stored frozen at-80℃prior to analysis.

The human GARP/TGF beta 1 complex protein is coated, and the concentration of each control antibody and each test antibody in serum is measured by adopting an indirect enzyme-linked immunosorbent assay. Pharmacokinetic parameters were calculated based on the blood concentration of each animal at different time points. See table 25 and fig. 25 for specific results.

Table 25 table of pharmacokinetic parameters of wild type C57 mice

Medicament	Half life, hours	Tmax(h)	Cmax(μg/mL)	AUClast(h*μg/mL)
h087-H2L2	340.78	0.25	218.42	39535.04
h171-H3L5	317.42	0.25	193.60	27633.70
h201-H5L3	244.35	0.25	243.22	39280.67
h195-H3L1	313.11	0.25	213.39	30637.56
h138-H8L5	359.52	0.25	234.28	30973.00
h017-H2dL2	366.90	0.25	245.26	35988.32
ARGX-115	332.93	0.25	233.56	43933.01
SRK-Ab6	39.98	0.25	242.46	18740.57

18.2 pharmacokinetic evaluation in human GARP Gene knock-in mice

The pharmacokinetic differences were compared using 9 SPF-grade female human GARP knock-in C57 mice, 6 weeks old, weighing about 19-21g, divided into 3 groups, each group being 3, and given a single tail vein injection of a 10mg/kg dose of control antibody (ARGX-115) and test antibody (H138-H8L 5 and H017-H2dL2 prepared in example 7.2).

The concentration of each control and test group drug in the present disclosure in serum was determined by indirect enzyme-linked immunosorbent assay, coating the human GARP/tgfβ1 complex protein. Pharmacokinetic parameters were calculated based on the blood concentration of each animal at different time points (table 26), see table 26 and fig. 26 for specific results.

TABLE 26 pharmacokinetic parameter Table for GARP transgenic mice

Medicament	Half life, hours	Tmax(h)	Cmax(μg/mL)	AUClast(h*μg/mL)
ARGX-115	344.14	0.25	207.35	32076.39
h017-H2dL2	257.40	0.25	248.99	33002.70
h138-H8L5	239.23	0.25	182.41	25996.83

Claims

An antibody or antigen binding fragment that binds to a GARP/tgfβ1 complex, wherein the antibody or antigen binding fragment comprises HCDR1-3, and/or LCDR1-3, the HCDR1-3 and/or the LCDR1-3 comprising a sequence shown in table 2 or table 15, respectively, or a sequence having at least 70% identity or at most 5 mutations to a sequence shown in table 2 or table 15;

Preferably, 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, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the HCDR2 comprises SEQ ID NO: 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 thereto or at most 5 mutations;

preferably, the HCDR3 comprises SEQ ID NO: 106. 111, 119, 124, 132, 137, 145, 150, 158, 163, 171, or 176, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the LCDR1 comprises SEQ ID NO: 112. 115, 125, 128, 138, 141, 151, 154, 164, 167, 177 or 180, or a sequence having at least 70% identity thereto or up to 5 mutations;

preferably, the LCDR2 comprises a sequence corresponding to SEQ ID NO: 113. 116, 126, 129, 139, 142, 152, 187, 155, 165, 168, 178 or 181, or a sequence having at least 70% identity thereto or at most 5 mutations;

Preferably, the LCDR3 comprises SEQ ID NO: 114. 127, 140, 153, 166, 179, or a sequence having at least 70% identity thereto or at most 5 mutations.
The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a sequence set forth in any one of groups (1) - (7):

(1) The HCDR1 comprises SEQ ID NO: 104. 107 or 109; the HCDR2 comprises SEQ ID NO: 105. 182-186, 108 or 110, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 106 or 111; and/or, the LCDR1 comprises SEQ ID NO:112 or 115, said LCDR2 comprises a sequence of any one of SEQ ID NOs 113 or 116, and said LCDR3 comprises SEQ ID NOs: 114;

(2) The HCDR1 comprises SEQ ID NO: 117. 120 or 122, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 118. 121 or 123, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 119 or 124; and/or, the LCDR1 comprises SEQ ID NO:125 or 128, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 126 or 129, and the LCDR3 comprises the sequence of any one of SEQ ID NOs: 127;

(3) The HCDR1 comprises SEQ ID NO: 130. 133 or 135, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 131. 134 or 136, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 132 or 137; and/or, the LCDR1 comprises SEQ ID NO:138 or 141, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 139 or 142, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 140;

(4) The HCDR1 comprises SEQ ID NO: 143. 146 or 148, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 144. 147 or 149, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 145 or 150; and/or, the LCDR1 comprises SEQ ID NO:151 or 154, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 152. 187 or 155, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 153;

(5) The HCDR1 comprises SEQ ID NO: 156. 159 or 161, said HCDR2 comprises the sequence of any one of SEQ ID NOs: 157. 188-189, 160 or 162, said HCDR3 comprises the sequence of any one of SEQ ID NOs: 158 or 163; and/or, the LCDR1 comprises SEQ ID NO:164 or 167, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 165 or 168, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 166;

(6) The HCDR1 comprises SEQ ID NO: 169. 172 or 174, and the HCDR2 comprises the sequence of any one of SEQ ID NOs: 170. 173 or 175, and the HCDR3 comprises the sequence of any one of SEQ ID NOs: 171 or 176; and/or, the LCDR1 comprises SEQ ID NO:177 or 180, and wherein said LCDR2 comprises the sequence of any one of SEQ ID NOs: 178 or 181, and wherein said LCDR3 comprises the sequence of any one of SEQ ID NOs: 179;

(7) Sequences having at least 70% identity or at most 5 mutations to each CDR in groups (1) - (6) HCDR1-3 and/or LCDR1-3;

preferably, the HCDR1-3 and/or the LCDR1-3 are determined according to Kabat, chothia or IMGT rules.
The antibody or antigen-binding fragment of claim 1 or 2, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the HCDR1-3 and/or a light chain variable region comprising the LCDR1-3;

preferably, the heavy chain variable region comprises: as set forth in 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 having at least 70% identity or up to 20 mutations to the sequence shown;

Preferably, the light chain variable region comprises: as set forth in 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, or a sequence having at least 70% identity or up to 20 mutations to the sequence shown;

more preferably, (1) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 15. 27, 32-40, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 16. 28-31;

more preferably, (2) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 17. 41, 46-48, and the light chain variable region comprises the sequence set forth in any one of SEQ ID NOs: 18. 42-45;

more preferably, (3) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 19. 49-51, 54-61, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 20. 52-53, 62-67;

more preferably, (4) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 21. 68, 70-72, and the light chain variable region comprises a sequence set forth in any one of SEQ ID NOs: 22. 69, 73-77;

more preferably, (5) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 23. 78, 81-87; the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO: 24. 79-80, 88-89;

More preferably, the heavy chain variable region of (6) comprises the amino acid sequence as set forth in SEQ ID NO: 25. 90, 93-99, and the light chain variable region comprises a sequence as set forth in any one of SEQ ID NOs: 26. 91-92, 100-103;

more preferably, (7) said heavy chain variable region and/or said light chain variable region comprises a sequence having at least 70% identity or up to 20 mutations to the sequences set forth in groups (1) - (6).
An antibody or antigen-binding fragment according to claim 3, wherein the heavy chain variable region and/or the light chain variable region is selected from VH and/or VL as set out in table 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 antibody or antigen binding fragment of any one of claims 1-3, wherein the at least 70% identity is preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; the up to 5 mutations are preferably up to 4, 3, 2, 1 or 0 mutations; the up to 20 mutations are preferably up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations; preferably, the mutation is an insertion, a deletion or a substitution, preferably a conservative amino acid substitution, preferably a back mutation or a hot spot mutation.
The antibody or antigen-binding fragment of any one of claims 1-5, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region and/or a light chain constant region;

preferably, the heavy chain constant region is selected from IgG, e.g., igG1, igG2, igG3 or IgG4; the IgG may be selected from human IgG, such as human IgG1 or human IgG4; the light chain constant region is selected from a kappa chain or a lambda chain, preferably a kappa chain;

more preferably, the heavy chain constant region comprises SEQ ID NO:9 or 10, and the light chain constant region comprises the sequence set forth in SEQ ID NO:12 or 13.
The antibody or antigen-binding fragment of any one of claims 1-6, wherein 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 (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, fab '-SH, F (ab') ₂ Fd, fv, scFv, diabodies (diabodies) or single domain antibodies.
The antibody or antigen-binding fragment of any one of claims 1-7, wherein the antibody or antigen-binding fragment further comprises a conjugate; the conjugate may be selected from a therapeutic agent, which may be selected from a radioisotope, a chemotherapeutic agent or an immunomodulator, or a tracer, which may be selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.
The antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment binds to a human GARP/tgfβ1 complex and/or a monkey GARP/tgfβ1 complex;

preferably, the antibody or antigen binding fragment binds to the human GARP/TGF-beta 1 complex with a KD value of less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M;

preferably, the antibody or antigen binding fragment binds to a monkey GARP/TGF-beta 1 complex with a KD value of less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M.
The antibody or antigen-binding fragment of any one of claims 1-9, wherein 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, not to (human) tgfβ1 monomer; (2) Binding to (human) TGF-beta 1 monomer, not to (human) GARP monomer, preferably having a KD value of less than 1E-7M, 1E-8M, 1E-9M, 1E-10M, 1E-11M, 1E-12M or 1E-13M binding to (human) TGF-beta 1 monomer; (3) Binding only the (human) GARP/tgfβ1 complex, not binding both the (human) GARP monomer and the (human) tgfβ1 monomer;

preferably, the binding site of the antibody or antigen binding fragment has a characteristic selected from one of the group consisting of: (1) the binding site is located in a (human) GARP monomer; (2) the binding site is located in a (human) tgfβ1 monomer; (3) The binding site is located on the (human) GARP/tgfβ1 complex and not only on GARP or tgfβ1 of said complex.
The antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment further has a characteristic selected from at least one of the following: (1) binding to a (human) GARP/tgfβ1 complex protein; (2) Binding to cells expressing the (human) GARP/tgfβ1 complex, e.g. (human) Treg cells; (3) blocking the formation of activated tgfβ1; (4) Inhibit (human) Treg cell function, e.g., inhibit SMAD2 protein phosphorylation.
A multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises at least a first antigen-binding moiety comprising the antibody or antigen-binding fragment of any one of claims 1-11, and a second antigen-binding moiety that binds to a different target than the first antigen-binding moiety or to a different epitope of the same target; preferably, the second antigen binding moiety is an antibody or antigen binding fragment;

preferably, the additional target is selected from the group consisting of: (1) Tumor Specific Antigens (TSA) or Tumor Associated Antigens (TAA); (2) an immune checkpoint; (3) recruiting and/or activating targets of immune cells.
A Chimeric Antigen Receptor (CAR), wherein the chimeric antigen receptor comprises at least an extracellular antigen binding domain comprising the antibody or antigen binding fragment of any one of claims 1-11 or the multispecific antigen-binding molecule of claim 12, a transmembrane domain, and an intracellular signaling domain.
An immune effector cell, wherein the immune effector cell expresses the chimeric antigen receptor of claim 13 and/or comprises a nucleic acid molecule encoding the chimeric antigen receptor of claim 13;

preferably, the immune effector cell is selected from T cells, NK cells (natural killer cell), NKT cells (natural killer T cell), 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;

preferably, the immune effector cell is an autoimmune effector cell or an alloimmune effector cell.
An isolated nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody or antigen binding fragment of claims 1-11, the multispecific antigen-binding molecule of claim 12, or the chimeric antigen receptor of claim 13.
A vector, wherein the vector comprises the nucleic acid molecule of claim 15.
A cell, wherein the cell comprises the vector of claim 16.
A method of making the antibody or antigen-binding fragment of claims 1-11, or the multispecific antigen-binding molecule of claim 12, wherein the method comprises: (1) Culturing the cell of claim 17 and/or (2) isolating the antibody or antigen-binding fragment expressed by the cell, or a multispecific antigen-binding molecule.
A method of making the immune effector cell of claim 14, wherein the method comprises introducing a nucleic acid molecule encoding the chimeric antigen receptor of claim 13 into the immune effector cell, and/or initiating expression of the chimeric antigen receptor by the immune effector cell.
A pharmaceutical composition comprising the antibody or antigen binding molecule of any one of claims 1-11, or the multispecific antigen binding molecule of claim 12, or the immune effector cell of claim 14, or the nucleic acid molecule of claim 15, or the vector of claim 16, or the cell of claim 17, or the product made according to the method of any one of claims 18-19; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant.
Use of the antibody or antigen binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the nucleic acid molecule of claim 15, the vector of claim 16, the cell of claim 17, the pharmaceutical composition of claim 20, or the product made according to the method of any one of claims 18-19, in the manufacture of a medicament for the treatment of a tgfβ -related disease; preferably, the tgfβ -related disease is selected from cancer, a fibrotic disease, inflammation, cardiovascular and cerebrovascular disease or chronic infectious disease.
A method of treating a tgfβ -related disease, wherein the method comprises administering to a subject an effective amount of a medicament comprising the antibody or antigen-binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the nucleic acid molecule of claim 15, the vector of claim 16, the cell of claim 17, the pharmaceutical composition of claim 20, or the product made according to the method of any one of claims 18-19, preferably the tgfβ -related disease is selected from cancer, a fibrotic disease, inflammation, a cardiovascular disease, or a chronic infectious disease.
The antibody or antigen binding fragment of any one of claims 1-11, the multispecific antigen-binding molecule of claim 12, the immune effector cell of claim 14, the nucleic acid molecule of claim 15, the vector of claim 16, the cell of claim 17, the pharmaceutical composition of claim 20, or the product made according to the method of any one of claims 18-19, wherein the medicament for treating a tgfp-related disease, preferably the tgfp-related disease is selected from cancer, a fibrotic disease, an inflammation, a cardiovascular disease, or a chronic infectious disease.