WO2022161425A1 - 抗tnfr2人源化抗体及其用途 - Google Patents

抗tnfr2人源化抗体及其用途 Download PDF

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WO2022161425A1
WO2022161425A1 PCT/CN2022/074228 CN2022074228W WO2022161425A1 WO 2022161425 A1 WO2022161425 A1 WO 2022161425A1 CN 2022074228 W CN2022074228 W CN 2022074228W WO 2022161425 A1 WO2022161425 A1 WO 2022161425A1
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antibody
seq
cells
antigen
tnfr2
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PCT/CN2022/074228
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English (en)
French (fr)
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赵晓峰
卢士强
曹卓晓
唐任宏
任晋生
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江苏先声药业有限公司
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Priority to KR1020237027516A priority Critical patent/KR20230132524A/ko
Priority to AU2022212842A priority patent/AU2022212842A1/en
Priority to US18/274,845 priority patent/US20240109974A1/en
Priority to EP22745298.4A priority patent/EP4286411A1/en
Priority to CA3206910A priority patent/CA3206910A1/en
Priority to JP2023546194A priority patent/JP2024504820A/ja
Priority to CN202280011902.8A priority patent/CN117120469A/zh
Publication of WO2022161425A1 publication Critical patent/WO2022161425A1/zh

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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C12N15/09—Recombinant DNA-technology
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/11—Antigen recognition domain
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to the fields of biomedicine and bioengineering, in particular, to humanized anti-TNFR2 antibodies or antigen-binding fragments thereof, as well as pharmaceutical compositions of the humanized anti-TNFR2 or antigen-binding fragments thereof, and uses thereof.
  • Immunity is a protective response of the body and is affected by many genes, proteins and cells. Immunity abnormalities can cause many diseases, including tumors, immune deficiencies (such as AIDS, etc.), allergies, and rheumatoid arthritis.
  • tumor immunotherapy as a new treatment method, has become a hot spot in the field of tumor treatment research.
  • Antagonistic antibodies targeting immune checkpoint proteins such as anti-PD-1 and anti-CTLA-4 mAbs, have been used to treat many types of cancer with revolutionary results, greatly prolonging the malignant tumor in patients lifetime.
  • many cancer patients still do not respond to treatment with antagonistic antibodies against immune checkpoint proteins or develop resistance or resistance after brief treatment. Therefore, it is necessary to develop new drugs for the treatment of cancer, which can be used alone or in combination with other tumor treatment methods, including the use of antagonistic antibodies against immune checkpoint proteins, to further improve the efficacy and safety.
  • the inventors have prepared an antagonistic antibody against TNFR2, which can 1) specifically bind to TNFR2, block the binding of TNFR2 and its ligand TNF ⁇ , thereby inhibit the proliferation of Treg cells and their mediated inhibitory function, and promote the effect of T cells.
  • the present invention prepares the anti-TNFR2 humanized antibody or its antigen-binding fragment, and prepares the anti-TNFR2 humanized or its antigen-binding fragment pharmaceutical composition, and verifies its use.
  • the present invention provides a humanized antibody or antigen-binding fragment thereof that specifically binds TNFR2, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region;
  • the heavy chain variable region comprises:
  • a. is selected from the VH sequence shown in any one of SEQ ID NO.19 ⁇ 27,
  • a. is selected from the VL sequence shown in any one of SEQ ID NO.28 ⁇ 37,
  • amino acid insertion, deletion and/or substitution occurs in the FR region of the heavy chain variable region or/and the light chain variable region;
  • substitutions are conservative amino acid substitutions.
  • the heavy chain variable region comprises CDRs that are completely identical to any of the sequences of SEQ ID NO. 19-27, and the FR has at least 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of VH sequences;
  • the light chain variable region comprises a Any sequence of SEQ ID NO. 28 to 37 is completely identical to the CDR, and the FR has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% VL sequences with % or 100% sequence identity.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region selected from the group consisting of:
  • (17) has VH shown in SEQ ID NO.27 and VL shown in SEQ ID NO.37;
  • the CDRs are completely identical to any one of the above-mentioned sequence combinations (1) to (17), and the FRs have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or 100% sequence identity of VH and VL combinations;
  • amino acid insertion, deletion and/or substitution occurs in the FR region of the heavy chain variable region or/and the light chain variable region;
  • substitutions are conservative amino acid substitutions.
  • the antibody or antigen-binding fragment thereof comprises: CDR1-VH is selected from SEQ ID NO. 1, 7, or 13; CDR2-VH is selected from SEQ ID NO. 2, 8, or 14; CDR3-VH is selected from SEQ ID NO. 3, 9, or 15; CDR1-VL is selected from SEQ ID NO. 4, 10, or 16; CDR2-VL is selected from SEQ ID NO. 5, 11, or 17; and CDR3-VL is selected from SEQ ID NO. 6, 12, or 18.
  • the antibody or antigen-binding fragment thereof of the present invention has a dissociation constant (KD) for binding to human TNFR2 not greater than 7 nM, and a dissociation constant (KD) for binding to cynomolgus monkey TNFR2 not greater than 5nM.
  • KD dissociation constant
  • KD dissociation constant
  • the antibody or antigen-binding fragment thereof of the invention comprises or does not comprise a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region comprises a full-length heavy chain constant region or a fragment thereof, which can be selected from a CH1 domain, an Fc domain or a CH3 domain; preferably, the heavy chain constant region and/or light chain constant region are human heavy chain constant regions and/or human light chain constant regions region; preferably, the heavy chain constant region may be selected from IgG heavy chain constant regions, such as IgG1 heavy chain constant regions, IgG2 heavy chain constant regions, IgG3 heavy chain constant regions or IgG4 heavy chain constant regions; preferably, the The heavy chain constant region is a human IgG1 heavy chain constant region, a human IgG2 heavy chain constant region, a human IgG3 heavy chain constant region, or a human IgG4 heavy chain constant region.
  • the antibodies or antigen-binding fragments thereof of the present invention are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (eg, bispecific antibodies) antibody), monovalent antibody, multivalent antibody, full length antibody, antibody fragment, Fab, Fab', F(ab')2, Fd, Fv, scFv, or diabody.
  • the antibody or antigen-binding fragment thereof of the present invention is coupled with another molecule, with or without a linker; preferably, the other molecule may be selected from a therapeutic agent or a tracer
  • the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents
  • the tracer agent is selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizer.
  • the antibody or antigen-binding fragment thereof of the present invention has the following properties:
  • the present invention provides a multispecific antigen-binding molecule comprising at least a first antigen-binding moiety and a second antigen-binding moiety, the first antigen-binding moiety comprising the present invention
  • the second antigen-binding moiety specifically binds to other antigens other than TNFR2 or binds to a different TNFR2 epitope from the first antigen-binding moiety;
  • the other antigens are selected from CD3 , CD4, CD5, CD8, CD14, CD15, CD16, CD16A, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD70, CD74, CD80, CD86 , CD126, CD138, B7, PD-1, PD-L1, PD-2, CTLA4, PRVIG, TIGHT, HAS, CLDN18.2, MSL
  • the present invention provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain; the cell
  • the outer antigen-binding domain comprises the TNFR2 humanized antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or comprises the multispecific antigen-binding molecule of the second aspect of the present invention.
  • the present invention provides an immune effector cell comprising the chimeric antigen receptor of the third aspect of the present invention or a nucleic acid encoding the chimeric antigen receptor of the third aspect of the present invention Fragments of ; preferably, the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer cells), monocytes, macrophages, dendritic cells or mast cells; the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells; preferably, the immune effector cells are allogeneic immune effector cells or autologous immune cells.
  • the present invention provides an isolated nucleic acid fragment encoding the TNFR2 humanized antibody or antigen-binding fragment thereof of the first aspect of the present invention, and the multispecific antigen-binding molecule of the second aspect of the present invention or the chimeric antigen receptor described in the third aspect.
  • the present invention provides a vector comprising the nucleic acid fragment of the fifth aspect.
  • the present invention provides a host cell comprising the vector of the sixth aspect; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast) ), insect cells or mammalian cells (CHO cell line or 293T cell line); preferably, the cells lack a fucosyltransferase, more preferably, the fucosyltransferase is FUT8.
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast) ), insect cells or mammalian cells (CHO cell line or 293T cell line); preferably, the cells lack a fucosyltransferase, more preferably, the fucosyltransferase is FUT8.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of any one of the present invention or the multispecific antigen-binding molecule of the present invention, the method comprising culturing the cell of the seventh aspect , and isolating the cell-expressed antibody or antigen-binding fragment or multispecific antigen-binding molecule thereof.
  • the present invention provides a method for preparing immune effector cells, the method comprising introducing a nucleic acid fragment encoding the aforementioned CAR into the immune effector cells, optionally, the method further comprising activating the immune effector cells express the CAR.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention, the multispecific antigen-binding molecule of the present invention, the chimeric molecule of the present invention antigen receptors, immune effector cells of the present invention, nucleic acid fragments of the present invention, vectors of the present invention, cells of the present invention, or products prepared by the methods of the present invention; preferably, the The composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the composition further comprises an additional anti-tumor agent, immunotherapeutic agent or immunosuppressive agent; preferably, the additional The anti-tumor agent is selected from PD-1 antibody, PD-L1 antibody or CTLA-4 antibody.
  • the present invention provides the antibody or antigen-binding fragment thereof, the multispecific antigen-binding molecule, the chimeric antigen receptor, the immune effector cell, and the nucleic acid.
  • the disease is a disease related to Treg cell and/or MDSC function; preferably, the disease is cancer or an autoimmune disease; preferably, the cancer is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic disease Colorectal cancer, triple negative breast cancer, pancreatic cancer, non-small cell lung cancer and/or advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma; preferably, the autoimmune disease It can be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuro
  • the present invention provides a method for treating and/or preventing an immune abnormality-related disease, the method comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof of the present invention, the The multispecific antigen-binding molecule, the chimeric antigen receptor, the immune effector cell, the nucleic acid fragment, the carrier, the cell, the product prepared by the method or the Pharmaceutical composition; preferably, the disease related to immune abnormality is a disease related to Treg cell and/or MDSC function; preferably, the disease is cancer or an autoimmune disease; preferably, the cancer is selected from ovarian cancer, advanced stage Epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastases melanoma; preferably, the autoimmune disease can be selected from rheumatoid arthritis
  • the present invention also provides administration of additional anti-tumor treatments, such as chemotherapeutic agents, targeted therapeutics, and immunotherapeutics, to the subject, including PD-1/PD-L1 treatments such as anti-PD-1/ PD-L1 antibody, anti-CTLA-4 therapeutic agent such as anti-CTLA-4 antibody; preferably, the additional anti-tumor therapy is selected from PD-1/PD-L1 therapy; preferably, the PD-1/PD- L1 therapy is selected from PD-L1 antibodies.
  • additional anti-tumor treatments such as chemotherapeutic agents, targeted therapeutics, and immunotherapeutics
  • the present invention provides an in vitro method for detecting TNFR2, comprising the step of contacting a sample suspected of containing TNFR2 with the antibody or antigen-binding fragment thereof of the present invention.
  • TNFR2 refers to tumor necrosis factor receptor 2, also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) or CD120b, a membrane that binds tumor necrosis factor-alpha (TNF ⁇ ) receptor.
  • TNFRSF1B tumor necrosis factor receptor superfamily member 1B
  • CD120b a membrane that binds tumor necrosis factor-alpha (TNF ⁇ ) receptor.
  • the TNFR2 is preferably human TNFR2.
  • anti-TNFR2 antibody refers to any immunoglobulin molecule comprising at least a portion capable of specifically binding TNFR2 (such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain chain or light chain variable regions, heavy or light chain constant regions, framework regions or any portion thereof) protein- or peptide-containing molecules.
  • CDR complementarity determining region
  • TNFR2 antibodies also include antibody-like protein scaffolds (eg, the tenth fibronectin type III domain (10Fn3)) that contain BC, DE, and FG structural loops that are similar in structure and solvent accessibility to the antibody CDRs.
  • the tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and by combining the residues of the BC, DE and FG loops of 10Fn3 with CDR-H1, CDR-H2 or CDRs from the TNFR2 monoclonal antibody - Residue substitutions in the H3 region, one skilled in the art can graft, eg, the CDRs of a TNFR2 monoclonal antibody onto a fibronectin scaffold.
  • antibody refers to an immunoglobulin molecule that specifically binds or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugated antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies), antibody conjugates) And antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab')2 fragments which lack Fc fragments of intact antibodies (cleared faster from animal circulation), thus lacking Fc-mediated effector functions (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated by reference) This article).
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) without limitation to the method by which the antibody is produced.
  • antigen-binding fragment and “antibody fragment” are used interchangeably and refer to one or more antibody fragments that retain the ability to specifically bind a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabodies, tribodies, affibodies, Nanobodies, aptamers or domain antibodies.
  • binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment comprising two Fab fragments connected at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of an antibody one-arm Constituent Fv fragments; (v) dAbs comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) consisting of VH or VL A dAb consisting of domains; (viii) discrete complementarity determining regions (CDRs); and (ix) a combination of two or more discrete CDRs, which may optionally be linked by synthetic link
  • the two domains of the Fv fragment, VL and VH are encoded by separate genes, the two domains can be joined using recombinant methods by a linker that enables it to be made in which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called a single-chain Fv (scFv); see, eg, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 , 1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • CDRs complementarity determining regions
  • FRs framework regions
  • amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within a variable domain can be considered as heterozygous hypervariable positions, as these positions can be considered to be within the variable hypervariable region under one set of criteria (such as IMGT or KABAT), while those within a different set of outside the variable hypervariable regions under standards such as KABAT or IMGT. One or more of these positions can also be found in extended hypervariable regions.
  • variable domains of native heavy and light chains each comprise four framework regions predominantly adopting a sheet configuration, which are connected by three CDRs (CDR1, CDR2 and CDR3) that form loops connecting the sheet structure , and in some cases form part of the lamellar structure.
  • CDR1, CDR2 and CDR3 The CDRs in each chain are held tightly together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, which constitute the heavy chain variable region (VH).
  • the CDR combination of the chain (or its variable region) (VHCDR combination);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the first CDR of the light chain variable region (VL), respectively
  • Three CDRs that make up the CDR combination of the light chain (or its variable region) (VLCDR combination).
  • Kabat numbering system generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , National Institutes of Health, Bethesda, Md., 1991).
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab.
  • VL refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv or Fab.
  • variable domains refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody
  • the variable domains have more conserved amino acid sequences.
  • a “heavy chain constant region” comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from a CH1, Fc or CH3 domain.
  • light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the papain hydrolyzed carboxy-terminal portion of an intact antibody, which typically comprises the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the EUKabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain, thus, the Fc region can include or excluding Lys447.
  • percent (%) sequence identity and “percent (%) sequence identity” are used interchangeably and refer to the alignment of sequences and the introduction of gaps (if necessary) to achieve maximum percent sequence identity ( For example, gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes) followed by amino acids (or nucleotides) of the candidate sequence. ) residues are identical in percentage to amino acid (or nucleotide) residues of the reference sequence. For purposes of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, eg, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence aligned for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence to 100% sequence identity.
  • the length of candidate sequences aligned for comparison purposes may be, for example, at least 30% (eg, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. .
  • amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids within each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions:
  • Acidic amino acids Asp(D) and Glu(E);
  • Non-polar uncharged amino acids Cys(C), Met(M) and Pro(P);
  • Aromatic amino acids Phe(F), Tyr(Y) and Trp(W).
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules such as antibodies or their antigens Binding fragment-specific recognition.
  • An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) for that particular antigen or epitope thereof.
  • Various immunoassay modalities can be used to select antibodies that specifically immunoreact with a particular protein or carbohydrate.
  • solid phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with proteins or carbohydrates.
  • multispecific antibody refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different antigen binding to different epitopes.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • the term “bispecific antibody” refers to an antibody, usually human or humanized, having monoclonal binding specificities for at least two different antigens.
  • one of the binding specificities can be detected against an epitope of TNFR2, the other can be against another epitope of TNFR2 or any other antigen, such as for cell surface proteins, receptors, receptor subunits , tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins or bacterial surface proteins are detected.
  • valency refers to the presence of a specified number of binding sites in an antibody/antigen binding molecule.
  • monovalent refers to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule. the existence of points.
  • humanized antibody refers to a genetically engineered, non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
  • Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.
  • chimeric antibody refers to an antibody having variable sequences of immunoglobulins derived from one source organism (eg, rat or mouse) and those derived from a different organism (eg, human). Constant regions of immunoglobulins.
  • Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, Bio Techniques 4: 214-221; Gillies et al, 1985 J Immunol Methods 125: 191-202; incorporated by reference above This article.
  • antibody conjugate refers to a conjugate/conjugate in which an antibody molecule is chemically bonded to another molecule, either directly or through a linker.
  • the other molecule may be a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutic agents or immunomodulatory agents, and the tracer is selected from radiographic contrast agents, paramagnetic ions, metals , fluorescent labels, chemiluminescent labels, ultrasound contrast agents or photosensitizers.
  • An “antibody conjugate” such as an antibody-drug conjugate (ADC), wherein the drug molecule is the other molecule in question.
  • ADC antibody-drug conjugate
  • antigen chimeric receptor herein refers to an artificial immune effector cell surface receptor engineered to be expressed on an immune effector cell and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, Examples are variable heavy or light chains of antibodies, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • Treg regulatory T cells
  • Treg also known as suppressor T cells
  • IL-12 are a group of lymphocytes that negatively regulate the body's immune response to maintain tolerance to self-antigens Sex, control the excessive immune response, avoid immune damage to normal cells, and prevent the occurrence of autoimmune diseases.
  • Tregs express the following biomarkers: CD4, FOXP3 and CD25 and are thought to be derived from the same lineage as naive CD47 cells. Treg plays an extremely important role in the occurrence of tumors. Many studies have shown that the number of Treg cells in the tumor microenvironment is significantly increased, including melanoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc.
  • Treg cells The number of cells is also closely related to the survival rate of tumor patients.
  • tumor cells will induce the proliferation of tumor-infiltrating Treg cells, and the proliferating Treg cells will massively secrete immunosuppressive factors such as TGF- ⁇ , inhibit the function of immune cells such as CD8+ T cells, and hinder the killing effect of immune cells on tumors.
  • immunosuppressive factors such as TGF- ⁇
  • immune cells such as CD8+ T cells
  • vector includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses, or other suitable replicons (eg, viral vectors).
  • DNA vectors eg, plasmids
  • RNA vectors eg. RNA vectors
  • viruses eg. viral vectors
  • viral vectors eg. viral vectors
  • Various vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
  • the expression vectors of the present invention contain polynucleotide sequences and additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences (eg, promoter and enhancer regions) that direct gene transcription.
  • Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on expression vectors.
  • Expression vectors of the present invention may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.
  • the terms "subject”, “subject” and “patient” refer to an organism receiving treatment for a particular disease or disorder (eg, cancer or infectious disease) as described herein.
  • subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, bovid family members (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
  • treatment refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) unwanted physiological changes or pathologies, such as cell proliferative disorders such as cancer, in the subject being treated or infectious disease).
  • beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
  • alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
  • the term "effective amount” refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue or subject, is effective to prevent or alleviate a disease condition or progression of the disease. "Effective amount” also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
  • a therapeutically effective dose refers solely to the amount of that ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
  • Figure 1 Shows the binding ability of the tested antibodies to human TNFR2 recombinant protein.
  • Figures A, B, and C show the binding ability of #001, #219, #224 tested antibody humanized molecules to human TNFR2 recombinant protein; WT is a human-mouse chimeric wild-type molecule, anti-chicken protein lysozyme (anti- Hel-WT) isotype control antibody was the negative control for this experiment.
  • Figure 2 Shows the binding ability of test antibodies to cynomolgus monkey TNFR2 recombinant protein.
  • Figures A, B and C are the binding ability of #001, #219, #224 tested antibody humanized molecules to cynomolgus monkey TNFR2 recombinant protein; WT is a human-mouse chimeric wild-type molecule, anti-chicken protein lysozyme ( anti-Hel-WT) isotype control antibody was the negative control for this experiment.
  • Figure 3 Shows the binding ability of the tested antibodies to CHO-TNFR2 cells highly expressing human TNFR2.
  • Figures A, B, and C show the binding ability of #001, #219, and #224 test antibody humanized molecules to CHO-TNFR2 cells, respectively; anti-Hel-WT is a negative isotype control antibody.
  • Figure 4 Shows the binding ability of the tested antibodies to Treg cells.
  • Panels A and B are the overlapping graphs of the binding of humanized molecules of #001 and #219 test antibodies to Treg, respectively.
  • the open peak is the binding map of anti-Hel-WT isotype negative control antibody
  • the solid peak is the binding map of each antibody to Treg cells; "only secondary antibody” and “unstained” (blank cell control without any antibody) All were negative controls, and the antibody concentration was 200ng/ml.
  • Figure 5 Shows the binding of test antibodies to CD8+ T cells in activated cynomolgus PBMC.
  • Panel A is an overlay of the binding of test antibodies to cynomolgus CD8+T at 2 ⁇ g/ml and 0.2 ⁇ g/ml, respectively.
  • Panel B shows the multiples of the average fluorescence intensity of the test antibody binding to cynomolgus monkey CD8+T at the concentrations of 2 ⁇ g/ml and 0.2 ⁇ g/ml respectively (the average fluorescence intensity of the test sample combined with the average fluorescence intensity of the sample with only the secondary antibody added) ratio).
  • the anti-hen protein lysozyme (anti-Hel) isotype control antibody was the negative control of this experiment.
  • Figure 6 Shows that the tested antibodies inhibit the interaction between human TNF[alpha] and human TNFR2 recombinant protein.
  • Figures A, B, and C show the inhibition of TNF ⁇ -TNFR2 by the humanized molecules of #001, #219, and #224, respectively; the anti-Helin-WT isotype control antibody was used in this experiment. negative control.
  • Figure 7 Shows that test antibodies inhibit the interaction of human TNF[alpha] with human TNFR2 expressed by CHO-TNFR2 cells.
  • Figures A, B, and C show the inhibition of the interaction between TNF ⁇ and CHO-TNFR2 by the humanized molecules of #001, #219, and #224, respectively; anti-Hel-WT is the negative isotype control antibody.
  • Figure 8. Shows the inhibitory effect of test antibodies on TNF ⁇ -induced degradation of I ⁇ B ⁇ .
  • Figure A is the gate-circle strategy for this experiment.
  • Panel B is the inhibitory effect of the test antibody on the degradation of I ⁇ B ⁇ caused by TNF ⁇ ;
  • the left panel is the histogram of the percentage of different concentrations of antibody and I ⁇ B ⁇ low ,
  • the right panel is the antibody concentration gradient on the percentage of I ⁇ B ⁇ low relative to the negative control antibody anti -Changes in multiples of Hel.
  • test concentrations of antibodies were 0.25, 2.5 and 25 ⁇ g/ml, respectively; “untreated Treg cells” refers to Treg cells without any treatment, TNF refers to Treg cells treated with 10ng/ml TNF ⁇ ; Rat IgG2b-PE is Negative isotype control (anti-Hel) for Anti-I ⁇ B ⁇ -PE antibody. Each concentration of the tested antibody was statistically different from the negative isotype control anti-Hel (**p ⁇ 0.01, ***p ⁇ 0.001, Two-Way ANOVA Analysis).
  • Figure 9 Shows inhibition of TNF ⁇ -induced Treg cell proliferation by test antibodies.
  • Panel A is an overlay of the effects of different test antibodies and negative isotype control anti-Hel on Treg proliferation, in which the dotted line is anti-Hel, and the solid line is the test antibody.
  • Panel B is the inhibitory effect of different antibodies on TNF ⁇ -induced Treg proliferation, the ordinate is the percentage of Treg proliferation, and the abscissa is the tested antibody.
  • "no drug treatment" that is, without any drug treatment
  • anti-Hel are the negative controls of the experimental system.
  • the experimental concentration of each antibody was 12.5 ⁇ g/ml.
  • Each tested antibody was statistically different from the negative isotype control anti-Hel (**p ⁇ 0.01, ***p ⁇ 0.001, One-Way ANOVA Analysis).
  • Figure 10 Shows the inhibitory effect of the test antibody on the inhibitory activity of Treg on the proliferation of Tcon cells.
  • Panel A is an overlay of the effect of different test antibodies and negative isotype control anti-Hel on Treg inhibition of Tcon cell proliferation, in which the dotted line is anti-Hel, and the solid line is the test antibody.
  • Figure B shows the inhibitory effect of different antibodies on Treg inhibiting the proliferation of Tcon cells, the ordinate is the percentage of Tcon cell proliferation, and the abscissa is the test antibody.
  • no drug treatment (i.e. Not treat) and Anti-Hel are the negative controls of the experimental system
  • Tcon+beads is the proliferation of Tcon in the absence of Treg cells, which is the positive control of the experimental system.
  • the experimental concentration of each antibody was 12.5 ⁇ g/ml.
  • Each tested antibody was statistically different from the negative isotype control anti-Hel (*p ⁇ 0.05, **p ⁇ 0.01, One-Way ANOVA Analysis).
  • FIG. 11 Shows ADCC killing effect of tested antibodies.
  • Panel A shows the ADCC killing effect of test antibodies on Treg.
  • Panel B shows the ADCC killing effect of the tested antibodies on CHO-TNFR2 cells highly expressing human TNFR2. Among them, anti-Hel is the negative control antibody.
  • Figures 12A-12C Shows expression identification of hTNFR2 and mTNFR2 in blood immune cells of hTNFR2 transgenic mice (TNFR2 HuGEMM).
  • Figure 12A shows the FACS gating strategy of immune cells in the blood of TNFR2 HuGEMM (also known as hTNFR2-GEMM) mice and the circled CD4+ T cells, CD8+ T cells and Treg cells on the expression levels and circles of mTNFR2 and hTNFR2 Gate's FMO control (Fluorescence Minus One control).
  • Figure 12B shows the peak graph of the expression levels of CD4+ T cells, CD8+ T cells and mTNFR2 and hTNFR2 in Treg cells in the blood of TNFR2 HuGEMM and Balb/c wild-type mice; dotted line: isotype, dashed line: BALB/c mice , solid line: TNFR2 HuGEMM.
  • Figure 12C is a statistical graph of the mean fluorescence intensity of mTNFR2 and hTNFR2 in CD4+ T cells, CD8+ T cells and Treg cells in the blood of TNFR2 HuGEMM and Balb/c wild-type mice.
  • Figures 13A-13C Show that TNFR2 antibodies inhibit mTNF[alpha]-induced Treg cell proliferation in TNFR2 HuGEMM (referred to as GEMM in the figure) mice.
  • Figure 13A shows the levels of human TNFR2 and mouse TNFR2 expressed by Treg cells in the spleen of GEMM mice and wild-type WT Balb/c mice.
  • Figures 13B and 13C show the effect of test TNFR2 antibody on mTNF ⁇ -induced Treg cell proliferation in GEMM mice or WT Balb/c mice without activator (B) or with activator (C) Dynabeads mouse T-activator CD3/CD28 inhibitory effect.
  • the experimental concentration of each antibody was 10 ⁇ g/ml.
  • Each antibody tested was statistically different from the negative isotype control (***p ⁇ 0.001, ****p ⁇ 0.0001, One-Way ANOVA Analysis).
  • Figure 14 Shows that anti-TNFR2 antibodies dose-dependently inhibit tumor growth in TNFR2 HuGEMM mice.
  • Figures A and B show that 219-Hu1-mIgG2a and 001-Hu3-mIgG2a can dose-dependently inhibit CT26-hTNFR2 KI at doses of 30 mg/kg, 10 mg/kg and 3 mg/kg compared with the vehicle control group. tumor growth.
  • Figure 15 Shows that anti-TNFR2 antibodies inhibit tumor growth in TNFR2 HuGEMM mice without causing weight loss in mice.
  • Figures A-B show the changes in absolute body weight of mice after administration of different doses of 219-Hu1-mIgG2a or 001-Hu3-mIgG2a.
  • Panels C-D show the change rate of the body weight of mice after administration of different doses of 219-Hu1-mIgG2a or 001-Hu3-mIgG2a.
  • Figure 16 Shows that anti-TNFR2 antibodies significantly inhibit tumor growth in two different PBMC donors in a human PBMC immunoreconstitution humanized mouse colon cancer tumor model.
  • Panel A shows that in donor #193, the anti-TNFR2 antibody 219-Hu1 has significant efficacy.
  • Panel B shows that in donor#272, on day 29 after tumor inoculation, tumor growth in 001-Hu3, 219-Hu1, and Keytruda groups was significantly inhibited compared with the control antibody group, and there were statistical differences (*** *: p ⁇ 0.0001, vs. isotype, Two way ANOVA).
  • Panels C-D show that in donor#193 and donor#272, mice in both control and administration groups experienced weight loss.
  • FIG. 17 Shows that anti-TNFR2 antibody can significantly increase the ratio of CD8+ T/Treg cells in tumor-infiltrating immune cells TIL.
  • Panel A shows a gating strategy for CD4+ T, CD8+ T and Treg cells in tumor-infiltrating TILs.
  • Panel B shows the ratio of CD4+T, CD8+T and Treg to CD45+ cells in tumor infiltrating leukocytes (TIL) after treatment with isotype antibody, 001-Hu3 antibody and 219-Hu1 antibody in donor#193.
  • Panel C shows the absolute numbers of CD4+T, CD8+T and Treg per mg of tumor tissue in TIL after treatment with isotype antibody, 001-Hu3 antibody and 219-Hu1 antibody in donor #193.
  • Panel D shows the ratio of CD8+ T/Treg in TIL after treatment with isotype antibody, 001-Hu3 antibody and 219-Hu1 antibody in donor #193.
  • Panel E shows the ratio of CD4+T, CD8+T and Treg to CD45+ cells in TIL after treatment with isotype antibody, 001-Hu3 antibody and 219-Hu1 antibody in donor#272.
  • Figure 17F shows the absolute numbers of CD4+T, CD8+T and Treg per gram of tumor tissue in TIL after treatment with isotype antibody, 001-Hu3 antibody and 219-Hu1 antibody in donor #272.
  • Figure 17G shows the ratio of CD8+ T/Treg in tumor-infiltrating TIL after treatment with isotype antibody, 001-Hu3 antibody and 219-Hu1 antibody in donor#272 (*: p ⁇ 0.05, **: p ⁇ 0.01, ** *: p ⁇ 0.001, unpaired T test).
  • Figure 18 Shows the tumor volume change curve of each treatment group in the hTNFR2 KI-CT26.1C03 colon cancer model.
  • FIG. 19 Shows the body weight change curve of each treatment group in the hTNFR2 KI-CT26.1C03 colon cancer model. This figure shows the change of absolute weight of mice after administration of different doses of 219-Hu1-hIgG1 antibody or combined with anti-mPD-L1 antibody.
  • Figure 20 Shows the body weight change rate curve of each treatment group in the hTNFR2 KI-CT26.1C03 colon cancer model. This figure shows the change rate of body weight of mice after administration of different doses of 219-Hu1-hIgG1 antibody or combined with anti-mPD-L1 antibody.
  • Figure 21 Shows the change in tumor body weight of each treatment group in the hTNFR2 KI-CT26.1C03 colon cancer model. This figure shows the changes in tumor weights at different doses of 219-Hu1-hIgG1 antibody or combined with anti-mPD-L1 antibody, isolated and weighed at the end of the experiment.
  • the "CDRs grafting" method is used for antibody humanization, that is, the human antibody with the highest homology is selected based on the sequence to provide antibody framework regions (FRs), and the antigen-binding fragment complementarity determining regions (CDRs) of the target antibody based on the Kabat nomenclature method are selected. ), grafted onto the former to form a humanized antibody variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Secondly, in order to effectively maintain antibody activity and affinity, based on antibody structure modeling (MOE software), select potential back mutation points, selection criteria: 1).
  • MOE software antibody structure modeling
  • the antigen-binding fragment complementarity determining regions (CDRs) of the Kabat nomenclature of the TNFR2 antibody are shown in Table 1
  • the VH and VL sequences of the humanized antibody molecules are shown in Table 2
  • the VH and VL sequences of the humanized anti-TNFR2 antibody are shown in Table 3.
  • VL sequence pairing situation is shown.
  • VL6 (SEQ ID NO.33) 224-Hu5 VH9 (SEQ ID NO. 27) VL9 (SEQ ID NO.36) 224-Hu6 VH8 (SEQ ID NO. 26) VL7 (SEQ ID NO.34) 224-Hu7 VH9 (SEQ ID NO. 27) VL10 (SEQ ID NO.37)
  • ExpiCHO-S cells were counted and seeded at a density of 2.5 to 4 ⁇ 10 6 cells/mL in freshly pre-warmed ExpiCHO expression medium (Invitrogen, A291002) for overnight culture.
  • the cell concentration is about 7-10 ⁇ 10 6 cells/mL, and the cell viability for transfection is greater than 95%.
  • the number of transfected cells take the required number of cells, dilute with ExpiCHO expression medium to a final concentration of 6 ⁇ 10 6 cells/mL, and place in a shaker at 37° C., 8% CO 2 , and 100 rpm for later use.
  • Preparation for transfection Dilute the plasmid in OptiPRO TM SFM medium (Invita, 12309019), and gently shake the centrifuge tube to mix.
  • the mixed ExpiFectamine TM CHO reagent (Inviscidgen, A29129) was added to the plasmid diluent, and the centrifuge tube was gently shaken to make it evenly mixed, and then allowed to stand at room temperature for 2 minutes.
  • the above-mentioned plasmid/ExpiFectamine TM CHO reagent complex was slowly dropped into the cell suspension to be transfected, and the flask was shaken during the dropping process.
  • the transfected cells were cultured in a shaker at a temperature of 37° C., 8% CO 2 and 100 rpm.
  • 0.6% ExpiFectamine TM CHO Enhancer Invidia, A29129
  • 16% ExpiCHO TM Feed Invidia, A29129
  • the flask was rotated and the cells were transferred to a shaker at 32°C, 5% CO 2 , and 100 rpm for culture.
  • 16% ExpiCHO TM Feed was supplemented to the transfected cells, and the flask was shaken gently during the addition.
  • the cell supernatant was collected and centrifuged at 4000g for 10 minutes to aspirate the supernatant, and the antibody was further purified.
  • the cell culture supernatant collected by high-speed centrifugation was filtered with a 0.45+0.22 ⁇ M filter membrane, and the first step was purified by affinity chromatography using Akta Avant 150 (Cytiva).
  • the chromatography medium was Mabselect PrismA (Cytiva, catalog number: 17549803), which interacts with Fc, and the equilibration buffer was PBS (2.5g/L Na 2 HPO 4 12H 2 O, 0.408g/L NaH 2 PO 4 , 8.76 g/L NaH 2 PO 4 , 8.76 g/L NaH 2 PO 4 g/L NaCl, pH 7.2), after equilibrating 4 times the column volume, the cell supernatant was loaded and combined, and the flow rate was controlled so that the retention time of the sample on the column was ⁇ 5min.
  • the column was rinsed with PBS (pH 7.2) until the A280 UV absorbance dropped to baseline. 2 column volumes were then rinsed with 20 mM PB + 1 M NaCl (3.752 g/L Na2HPO4 ⁇ 12H2O, 1.314 g/L NaH2PO4 , 58.44 g/L NaCl pH6.2). The column was then rinsed with PBS (pH 7.2) until the A280 UV absorbance and conductance reached baseline. Finally, the column was washed with the elution buffer of 20mM citric acid (2.184g/L citric acid, 1.086g/L sodium citrate, pH 3.4), and the elution peaks were collected according to the A280 UV absorption peak.
  • Example 2 ELISA detects the specific binding of TNFR2 humanized antibody to human and cynomolgus monkey TNFR2 protein
  • the binding results of the anti-TNFR2 antibody with human TNFR2 protein are shown in Figure 1 and Table 4, and the binding results with cynomolgus monkey TNFR2 are shown in Figure 2 and Table 4.
  • the data show that all the tested antibodies can specifically bind to human or monkey TNFR2 protein, and the binding ability of the humanized antibody and the human-mouse chimeric antibody is basically the same.
  • Example 3 BIAcore detects the affinity of TNFR2 humanized antibody to human and cynomolgus monkey TNFR2 protein
  • the antibody was injected and then a gradient concentration of TNFR2 protein was injected to make the antigen and antibody bind and dissociate.
  • Regeneration of the Protein A chip was performed with Glycine pH 1.5 after each cycle.
  • the affinity KD of the antibody antigen was fitted by BIAcore T200 analysis software. From the results in Table 5, it can be seen that all the tested anti-TNFR2 antibodies have specific binding with human or cynomolgus monkey TNFR2 protein, and the affinity level is high.
  • Example 4 FACS detects the binding of TNFR2 antibody to human TNFR2 on the surface of CHO-TNFR2 cells
  • CHO-K1 stable cells (named CHO-TNFR2) transfected with a high-expression plasmid of human TNFR2 were taken.
  • the transfected full-length plasmid of human TNFR2 was purchased from Sino Biological (Cat. No.: HG10417-UT, NCBI Ref Seq: NM_001066.2) , experiments were performed when the cell density did not exceed 80%.
  • the cell culture medium was discarded, rinsed with PBS, and digested with 1 ml of trypsin for 2 minutes. The digestion was terminated with Ham's F12 complete medium containing 10% FBS to prepare a cell suspension.
  • Dilute PE goat anti-Human IgG Fc antibody (ebioscience, product number: 12-4998-82) 250 times with staining buffer, add 100 ⁇ l per well to the washed cell wells, mix well, and stain at 4°C for 30 minute. After staining, the cells were also washed twice with staining buffer, and finally the cells were resuspended with 200 ⁇ l of staining buffer, and the signal (Thermo Attune NxT) was detected by flow cytometry. The stronger the signal, the stronger the binding ability of the antibody to TNFR2.
  • the binding curve of the antibody was drawn, and the four-parameter fitting (GraphPad Prism9) was used to calculate the EC50 value.
  • the smaller the EC50 value the stronger the ability of the antibody to bind to CHO-TNFR2 cells.
  • the binding effect of the humanized antibody was normalized to its corresponding human-mouse chimeric WT antibody. The higher the percentage value, the better the binding effect of the humanized antibody.
  • all the tested anti-TNFR2 antibodies can bind to human TNFR2 on the surface of CHO-TNFR2 cells, and the binding ability of the humanized antibody and the human-mouse chimeric WT antibody is basically the same.
  • Human Treg cells were isolated from human PBMC using a sorting kit (Stemcell, Cat. No.: 18063), and were obtained after 15 days of in vitro stimulation and expansion with Dynabeads Human Treg Expander (Gibco, Cat. No.: 11129D) and frozen in aliquots.
  • the Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300 ⁇ g for 5 minutes the next day, resuspended in DPBS to obtain a cell suspension, and counted.
  • the number of cells required for the experiment was added to a centrifuge tube, centrifuged at 300 ⁇ g for 5 min, the supernatant was removed, the cell density was adjusted to 2 ⁇ 10 6 /ml with staining buffer, and plated in a 96-well plate with 50 ⁇ l per well. Take all test antibodies and control antibody anti-Hel isotype (diluted to 200ng/ml with PBS, the total amount is 100 ⁇ l). Each sample was added to each well, 50 ⁇ l per well. Place the well plate with the cell suspension and the antibody on a microplate shaker, shake at 500 rpm for 1 min, and mix the cells and the antibody well. After mixing, the plate was placed in a refrigerator at 4°C and incubated for 60 min.
  • staining buffer After incubation, add 100 ⁇ l of staining buffer to each well, centrifuge at 350 ⁇ g for 5 min, and discard the supernatant; add 200 ⁇ l of staining buffer to each well to resuspend the cells, centrifuge at 350 ⁇ g for 5 min, and discard the supernatant.
  • staining buffer fluorescent staining antibody
  • cryopreserved cynomolgus monkey PMBC cells were recovered, they were resuspended in complete medium (RPMI1640-Glutamax+10% FBS+1 ⁇ P/S+1 ⁇ ITS+50 ⁇ MPM mercaptoethanol) to obtain a cell suspension and counted. According to the counting results, resuspend the cells at a density of 5 ⁇ 10 6 /ml, and spread 100 ⁇ l of the cell suspension per well in a 96-well U-bottom plate.
  • the activated cells were collected and counted, the cell density was adjusted to 4 ⁇ 10 6 /ml with staining buffer (2% FBS in DPBS), and 96-well V-bottom plates were plated with 50 ⁇ l per well.
  • test antibodies and anti-Hel control antibody (diluted to 0.4 ⁇ g/ml and 4 ⁇ g/ml with staining buffer, respectively) were taken, and each sample was added to each well, 50 ⁇ l per well. Place the well plate with the cell suspension and the antibody on a microplate shaker, shake at 500 rpm for 1 min, and mix the cells and the antibody well. After mixing, the plate was placed in a 4°C refrigerator and incubated for 30 min.
  • FIG. 5 shows that the Anti-Hel negative isotype control antibody does not bind to cynomolgus monkey CD8 T cells, and all tested anti-TNFR2 humanized antibodies can effectively bind to CD8+ T cells in activated cynomolgus monkey PBMC.
  • Example 7 ELISA detects that anti-TNFR2 antibody blocks the binding of TNF ⁇ and TNFR2
  • the microtiter plate was pre-coated with 1 ⁇ g/ml 100 ⁇ l/well human TNFR2 (Novoprotein, Cat. No. C830), and all the tested TNFR2 antibodies were diluted 2.5 times from the highest concentration of 30 ⁇ g/ml, and the diluted antibodies were respectively mixed with 15ng/ml Human TNF ⁇ -Biotin (Acro Biosystem, Cat. No.
  • TNA-H82E3 was mixed in equal volume, added to the ELISA plate at 100 ⁇ l/well, and incubated with shaking at room temperature for 2.0 hours; after washing the plate, added Streptavidin-HRP working solution (1:10000 dilution), 100 ⁇ l /well, incubate with shaking at room temperature for 40 minutes; wash the plate again, add HRP substrate TMB for color development, add stop solution to stop the reaction, and read the absorbance value with a microplate reader (MDI3). Taking the antibody concentration as the abscissa and the corresponding OD value as the ordinate, the inhibition curve of the antibody was drawn, and the four-parameter fitting (GraphPad Prism9) was used to calculate the IC50 value.
  • the blocking effect of the humanized antibody was normalized to its corresponding human-mouse chimeric antibody WT. The higher the percentage value, the better the inhibitory effect of the humanized antibody.
  • the blocking curve of the tested antibody is shown in Figure 6, and the inhibitory activity is shown in Table 7. It can be seen from Figure 6 and Table 7 that all the tested antibodies can significantly inhibit the binding of human TNF ⁇ and human TNFR2 protein, and the inhibition ability of the humanized antibody and the human-mouse chimeric WT antibody is basically the same.
  • Test antibodies block binding of TNF ⁇ and TNFR2
  • Example 8 Anti-TNFR2 antibody blocks the binding of TNFR2 to TNF ⁇ on CHO cells overexpressing human TNFR2
  • CHO-TNFR2 cells were digested, washed twice with DPBS, stained with Live/Dead (L/D) (20 minutes at room temperature), and plated at 1 ⁇ 10 5 cells/50 ⁇ l/well; the test anti-TNFR2 antibody was stained with The buffer was diluted to 6 ⁇ g/ml as the starting concentration, 3.33-fold gradient dilution, a total of 7 concentration points; 50 ⁇ l/well of the diluted antibody was added to the plated cells, mixed by gentle pipetting, and incubated at 4°C for 30 minutes.
  • L/D Live/Dead
  • the inhibition curve of the antibody was drawn, and the four-parameter fitting (GraphPad Prism9) was used to calculate the IC50 value.
  • the smaller the IC50 value the stronger the ability of the antibody to inhibit the binding of human TNF ⁇ to human TNFR2.
  • the blocking effect of the humanized antibody was normalized to its corresponding human-mouse chimeric WT antibody. The higher the percentage value, the better the inhibitory effect of the humanized antibody.
  • the blocking curve of the tested antibody is shown in Figure 7, and the inhibitory activity is shown in Table 8. It can be seen from Figure 7 and Table 8 that all the tested antibodies can significantly inhibit the binding of TNF ⁇ to TNFR2 on CHO-TNFR2 cells.
  • TNFR2 antibodies block binding of TNF ⁇ to CHO cell surface TNFR2
  • Example 9 FACS detection antibody inhibits TNF ⁇ -induced degradation of human Treg cells I ⁇ B ⁇
  • Human Treg cells were isolated from human PBMCs using a sorting kit (Stemcell, Cat. No.: 18063), stimulated and expanded in vitro with Dynabeads Human Treg Expander (Gibco, Cat. No.: 11129D) for 15 days, and then were obtained in aliquots and frozen.
  • the Treg cells isolated and expanded in vitro were recovered overnight, centrifuged at 300 ⁇ g for 5 minutes on the next day, and counted with AIM-V medium (brand Gibco, product number 31035025), and adjusted the cell density to 6 ⁇ 10 6 cells according to the cell counting results. Cells/ml, plated in 96-well plates, 25 ⁇ l per well.
  • Example 10 TNFR2 antibody inhibits Treg cell proliferation induced by TNF ⁇ and IL-2
  • Treg cells induced by TNF ⁇ and IL-2 By measuring the proliferation of Treg cells induced by TNF ⁇ and IL-2 in the presence of TNFR2 antibody, the inhibitory effect of the antibody on the proliferation of Treg cells induced by TNF ⁇ and IL-2 was determined (Zaragoza B et al., Nat Med. 2016Jan; 22(1) :16-7).
  • the cryopreserved Treg cells after in vitro expansion and separation were recovered overnight, centrifuged at 400 ⁇ g for 5 minutes the next day, and resuspended in medium to obtain a cell suspension and counted.
  • Treg was stained with CellTrace Violet cell proliferation kit (Thermo product number C34557), using 1 ⁇ l of stock solution per 1 ⁇ 10 7 cells to make 1 ml of staining solution.
  • the inhibitory effect of the antibody on the inhibitory function of Treg cells was determined by measuring the inhibitory function of Treg cells on responder T cells under the condition of containing anti-TNFR2 antibody. Take Treg (CD4+CD25+FoxP3+T cells) and Conventional T cell (Tcon) effector cells (CD4+CD25-T cells) required for the experiment and recover overnight, centrifuge at 400 ⁇ g for 5 minutes the next day, and use complete medium (RPMI1640-Glutamax+10%FBS+1 ⁇ P/S+1 ⁇ ITS+50 ⁇ MPM mercaptoethanol) to obtain cell suspension and count.
  • Treg CD4+CD25+FoxP3+T cells
  • Tcon Conventional T cell effector cells
  • Tcon cells were stained with CellTrace Violet cell proliferation kit (Thermo product number C34557), using 1 ⁇ l of stock solution per 1 ⁇ 10 7 cells to make 1 ml of staining solution. Stain at 37°C for 20 min, neutralize the reaction with at least 5 times the volume of complete medium, let stand for 5 min, and centrifuge at 400 ⁇ g for 5 min. Cells were washed once by resuspending in medium, and the supernatant was discarded by centrifugation again.
  • CellTrace Violet cell proliferation kit Thermo product number C34557
  • Treg cells were prepared, and Treg cells and Tcon cells were co-cultured at a ratio of 1:1.
  • the corresponding anti-CD3/CD28 Dynabeads (Gibco, Cat. No. 11129D) were prepared at a ratio of 1/10 according to the number of Tcon cells.
  • a volume of 50 ⁇ l per well was added to the medium of Treg and Tcon.
  • the antibody to be tested was added to each well, and the final concentration of the antibody was 12.5 ⁇ g/ml.
  • Example 12 FACS detection of anti-TNFR2 antibody-mediated antibody-dependent cytotoxicity (ADCC) activity
  • PBMC and cryopreserved Treg cells were cultured in complete medium (RPMI1640-Glutamax+10%FBS+1 ⁇ P/S+1 ⁇ ITS+50 ⁇ MPM mercaptoethanol), in which PBMC needed to add 100IU/ml IL-2 .
  • NK cells were separated from PBMC according to the requirements of the separation kit (Stemcell, Cat. No. 17955), and resuspended in complete medium (without IL-2) at a density of 0.8 ⁇ 10 6 /ml.
  • the target cells Treg cells or CHO-TNFR2 cells were labeled with Cell Trace violet reagent, and the density was adjusted to 0.4 ⁇ 10 6 /ml after labeling.
  • the antibody to be tested was serially diluted to 4 times the final concentration with complete medium, and set aside.
  • the target cells were seeded into the cell plate according to the distribution diagram of the experimental plate, 50 ⁇ l per well, and then the diluted drug was spread into the corresponding wells and incubated with the target cells at 37°C for 30 minutes. After the incubation, 100 ⁇ l of effector cell suspension was added to the wells to which effector cells were added, and 100 ⁇ l of culture medium was added to the unneeded wells, and incubated at 37° C. for 4 h. After the reaction, 1 ⁇ l of PI dye was added to each well and detected on the machine. The higher the positive ratio of PI in the target cells, the more significant the ADCC effect is. The results of the ADCC experiment are shown in Figure 11. The anti-Hel negative control antibody did not show ADCC killing effect because it had no target; the three tested humanized antibodies all showed ADCC activity on target cells Treg or CHO-TNFR2 cells effect.
  • the hTNFRSF1B-WPRE-PA expression cassette was knocked into the exon2 site of the Tnfrsf1b gene, and the heterozygous BALB/c mice with the Tnfrsf1b gene knocked into hTNFRSF1B-WPRE-PA were obtained.
  • Homozygous transgenic mice TNFR2 HuGEMM
  • TNFR2 HuGEMM expressing the human Tnfrsf1b (TNFR2) gene were then obtained by breeding.
  • mice from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • TNFR2 HuGEMM mice were collected and treated with ACK (Gibco, Cat. No. A1049201) to remove red blood cells for FACS immunophenotyping , to detect the expression of mTNFR2 and hTNFR2 on CD4+T cells, CD8+T cells and CD4+CD25+Foxp3+ Treg cells in peripheral blood of mice, all cell populations were gated based on FMO control ( Figure 12A).
  • TNFR2 HuGEMM mice did not express mTNFR2, and the expression profile/expression level of hTNFR2 on the surface of CD4+ T cells, CD8+ T cells and Treg cells was consistent with the expression profile/expression level of mTNFR2 on BALB/c background mice, indicating that TNFR2 HuGEMM Mice completed the replacement of mTNFR2 by hTNFR2 ( Figures 12B-12C).
  • TNFR2 HuGEMM mice By measuring the proliferation of Treg cells in TNFR2 HuGEMM mice induced by TNF ⁇ in the presence of TNFR2 antibody, it was verified that the TNFR2 signaling pathway and corresponding functions were preserved after genetic modification of TNFR2 HuGEMM mice, ensuring that the modified transgenic mice can be used for candidate anti-TNFR2 antibodies In vivo efficacy evaluation. Take 5 TNFR2 HuGEMM mice and 5 wild-type Balb/c mice, take out the mouse spleen aseptically in a biological safety cabinet for grinding, transfer all the ground cell suspensions to a 15 ml centrifuge tube, centrifuge (320 g) , 7 min, 4°C). Pour off the supernatant.
  • the sorted Treg cells were stained with the CellTrace Violet cell proliferation kit (Thermo product number C34557), and 1 ⁇ l of the storage solution was used for each 1 ⁇ 10 7 cells to prepare 1 ml of staining solution. Stain at 37°C for 20 min, neutralize the reaction with at least 5 times the volume of complete medium, let stand for 5 min, and centrifuge at 400 ⁇ g for 5 min. Cells were washed once by resuspending in medium, and the supernatant was collected by centrifugation again.
  • Thermo product number C34557 CellTrace Violet cell proliferation kit
  • test antibody including three humanized TNFR2 antibodies, one anti-mouse TNFR2 surrogate antibody Surrogate antibody clone #75-54.7 (BioXcell, BE0247) and the isotype control antibody anti-Hel-mIgG1 to each well, and the final concentration of the antibody is 10 ⁇ g/ml, incubate for 30 minutes.
  • mice IL-2 brand Yiqiao Shenzhou, product number 51061-MNAE, final concentration 300IU
  • mouse TNF ⁇ brand Yiqiao Shenzhou, product number 20180502, final concentration 50ng/ml
  • 50 ⁇ l of medium without or with Dynabeads mouse T-activator CD3/CD28 Gibco, Cat. No. 11452D, the ratio of beads to Treg cells is 1:20
  • the final volume of each well is 200 ⁇ l.
  • control wells with Treg cells added only with mouse IL-2, Treg cells only with mouse TNF ⁇ , and Treg cells added with mouse IL-2 and human TNF ⁇ were set.
  • FIG. 13A shows that Treg cells from TNFR2 HuGEMM mice express human TNFR2 but not murine TNFR2; Treg cells from WT Balb/c mice express only murine TNFR2.
  • Figures 13B and 13C show that, regardless of whether the Dynabeads mouse T-activator CD3/CD28 agonist was in the system, the proliferation of TNFR2 HuGEMM Tregs increased significantly when mIL-2 and mTNF ⁇ or hTNF ⁇ were added, indicating that TNFR2 HuGEMM mice were genetically modified with TNFR2 mediated signals are preserved.
  • the proliferation of WT balb/c was only increased under the addition of mIL-2 and mTNF ⁇ , while hTNF ⁇ did not affect the proliferation, indicating that hTNF ⁇ did not bind to mTNFR2.
  • Anti-TNFR2 humanized antibody was added to TNFR2 HuGEMM Treg in the presence of mIL-2 and mTNF ⁇ .
  • 001-Hu3-mIgG1 and 219-Hu1-mIgG1 could significantly inhibit the proliferation of TNFR2 HuGEMM Treg, And there was a statistical difference (***p ⁇ 0.001), while the anti-mouse TNFR2 surrogate antibody TR75-54.7 had no inhibitory effect; for WT Balb/c Treg, adding anti-TNFR2 antibody in the presence of mIL-2 and mTNF ⁇ , compared with the negative control Hamster Compared with IgG, anti-mouse TNFR2 surrogate antibody TR75-54.7 can significantly inhibit the proliferation of WT Balb/c Treg, and there is a statistical difference (***p ⁇ 0.001), while 001-Hu3-mIgG1 and 219-Hu1-mIgG1 both No inhibitory effect.
  • the EGE system based on CRISPR/Cas9 developed by Biositu Gene Biotechnology Co., Ltd. was used to replace part of Exon2 and Exon3 of the mTNFR2 gene in the mouse CT26 cell line with PuroR cassette, so as to achieve the purpose of gene knockout and destroy the mouse Expression of endogenous mTNFR2, then insert CDS of mouse intracellular domain, transmembrane domain and human extracellular domain chimeric protein to prepare CT26 cell line (CT26-hTNFR2 KI) expressing only hTNFR2 gene.
  • CT26-hTNFR2 KI CT26-hTNFR2 KI
  • the colon cancer animal model of CT26-hTNFR2 KI cell line with knock-in hTNFR2 gene was established by TNFR2 HuGEMM, and the efficacy of anti-TNFR2 antibody was tested.
  • each dose group of the tested anti-TNFR2 antibody group was statistically different from the PBS control group, and the drug effect was obvious; and the same antibody between different dose groups
  • the efficacy of the positive drug anti-mPD-1 BioXcell, BE0146
  • the tested anti-TNFR2 antibodies 219-Hu1-mIgG2a and 001-Hu3-mIgG2a at the dose of 3 mg/kg.
  • the pharmacodynamics were comparable, and the above results indicated that the tested anti-TNFR2 humanized antibodies had significant in vivo tumor growth inhibitory efficacy in a dose-dependent manner (A and B in Figure 14, Table 10).
  • mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • 7 ⁇ 10 6 people were intraperitoneally injected on the same day.
  • Peripheral blood mononuclear cells PBMC, donor#193 and donor#272, extracted from the whole blood of selected donors by Crown Biotechnology (Taicang) Co., Ltd. according to SOP-CP-042
  • PBMC Peripheral blood mononuclear cells
  • mice with moderate tumor volume were selected into the group, and the animals were divided into groups according to the tumor volume using Study DirectorTM (version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA) were randomly divided into 5 experimental groups (G1 is PBS control group, G2 is control antibody anti-Hel hIgG1 group, G3 is anti-TNFR2 antibody 001-Hu3-hIgG1 group, G4 is anti-TNFR2 antibody 219-Hu1- hIgG1 group and G5 are Keytruda group), 5-7 donors in each group, used for in vivo pharmacodynamics study. Dosing began on the day of the group (day 13).
  • the administered doses were all 10 mg/kg (same meaning as the measurement unit mpk in the figure, the same below), administered by intraperitoneal injection twice a week.
  • Tumor volume and body weight were measured twice a week at fixed times.
  • the tumor growth of the 219-Hu1 group was significantly inhibited compared with the control antibody group, and there was a statistical difference (P ⁇ 0.0001), and the other groups were compared with the control antibody group No statistical difference.
  • mice in both the control group and the administration group lost weight, and all of them died.
  • the veterinary autopsy results speculated that the death of the mice was caused by host immune rejection (GvHD) after the injection of PBMC.
  • GvHD host immune rejection
  • the test drugs 001-Hu3, 219-Hu1 and the positive drug Keytruda showed no obvious drug toxicity, and the treatment could be tolerated (C-D in Figure 16).
  • mice in the G2 control antibody group, the G3 anti-TNFR2 antibody 001-Hu3 group, and the G4 anti-TNFR2 antibody 219-Hu1 group were selected for tumor-infiltrating immune cell (TIL) isolation (Miltenyi, Cat. No. 130). -095-929), immunophenotyping by FACS, and adding 123 count eBeads (eBioscience, Cat. No. 01-1234-42) for absolute cell count, all cell populations were gated based on FMO control (A in Figure 17) .
  • TIL tumor-infiltrating immune cell
  • Example 17 Efficacy detection of the combination of 219-Hu1-hIgG1 monoclonal antibody and anti-mouse PD-L1 monoclonal antibody in TNFR2 humanized BALBc mouse model
  • mice with moderate tumor volume were selected into the group, and the animals were randomly assigned to 5 experimental groups (G1 for the control group, G2 for the anti- -mPD-L1 10mg/kg antibody group, G3 is 219-Hu1-hIgG1 10mg/kg antibody group, G4 is 219-Hu1-hIgG1 30mg/kg antibody group, G5 is 219-Hu1-hIgG1 10mg/kg+anti-mPD -L1 10mg/kg antibody combination group), 10 animals in each group were used for in vivo pharmacodynamic study, and the grouping (the 10th day) started administration on the same day. It was administered by intraperitoneal injection twice a week. Tumor volume and body weight were measured three times a week at fixed times.
  • the G2 anti-mPD-L1 10 mg/kg group On the 18th day after tumor inoculation (ie, the 8th day of administration), compared with the control group (G1), the G2 anti-mPD-L1 10 mg/kg group, the G3 219-Hu1-hIgG1 10 mg/kg group and the G4 219-
  • Relative tumor growth rate T/C% T RTV /C RTV ⁇ 100% (T RTV : mean RTV of the treatment group; C RTV : mean RTV of the negative control group).
  • TGI TV (%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100% calculation
  • Ti the mean tumor volume of the treatment group on the ith day of administration
  • T0 the treatment group after the administration
  • Vi the mean tumor volume of the solvent control group on the ith day of administration
  • V0 the mean tumor volume of the solvent control group on the 0th day of administration
  • the tumor weight of 219-Hu1-hIgG1 at two doses had a dose
  • TGI Tw (%) [1-Ti/Vi] ⁇ 100% calculation (Ti: the mean tumor weight of the treatment group on the ith day of administration, Vi: the mean tumor weight of the solvent control group on the ith day of administration ).

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Abstract

本发明公开了一种能够特异性地结合TNFR2的人源化抗体或其抗原结合片段，所述人源化抗体或其抗原结合片段能够调节免疫细胞的功能，可作为药物治疗与免疫异常有关的疾病，例如肿瘤。

Description

抗TNFR2人源化抗体及其用途
本申请要求于2021年1月29日提交到中国国家知识产权局的发明名称为“抗TNFR2人源化抗体及其用途”的中国专利申请202110140980.5和于2021年8月31日提交到中国国家知识产权局的发明名称为“抗TNFR2人源化抗体及其用途”的中国专利申请202111016307.7的优先权，其内容均通过引用以整体并入本文。
技术领域
本发明涉及生物医药和生物工程领域，具体而言，涉及抗TNFR2的人源化抗体或其抗原结合片段，以及所述抗TNFR2人源化或其抗原结合片段的药物组合物及其用途。
背景技术
免疫是机体的一种保护反应，受到很多基因、蛋白质以及细胞的作用。免疫异常会引起许多疾病，包含肿瘤、免疫缺陷(如艾滋病等)、过敏和类风湿性关节炎等疾病。在过去几年中，肿瘤免疫疗法作为一种全新的治疗方式，已成为肿瘤治疗研究领域的一大热点。靶向免疫检查点蛋白的拮抗性抗体，如抗PD-1和抗CTLA-4单抗，已被用于治疗多种类型的癌症，且取得了革命性的结果，大大延长了恶行肿瘤患者的生存期。然而还是有许多癌症患者对于免疫检查点蛋白的拮抗性抗体的治疗没有反应或在短暂治疗后产生抗性或耐药性。因此需要开发新的治疗癌症的药物，可单用或与其它肿瘤治疗方法联用，包含与免疫检查点蛋白的拮抗性抗体联合使用，以进一步提高疗效和安全性。
发明人发现，人TNFR2在人Treg和人的多种肿瘤细胞表面过度表达，提示人TNFR2可能促进患者肿瘤发生并介导肿瘤微环境的免疫抑制和免疫逃逸。通过TNFR2调控Treg细胞的功能，从而抑制肿瘤的发生，可能是一条非常有潜力的抗肿瘤策略。发明人制备了针对TNFR2的拮抗性抗体，其可以1)特异性结合TNFR2，阻断TNFR2和其配体TNFα的结合，从而抑制Treg细胞的增殖及其介导的抑制功能，促进效应T细胞的扩增以及效应T细胞和其他免疫细胞介导的抗肿瘤功能；2)由于TNFR2在人的肿瘤细胞系上高表达，此抗体也可直接介导对TNFR2高表达肿瘤细胞的杀伤作用；3)和现有抗PD-1/L1联用有更好的抗肿瘤效果并形成持久的免疫记忆。针对上述抗TNFR2的拮抗性抗体的描述，请参见PCT/CN2020/106057，该专利申请的全部内容通过引用的方式结合于本申请中。
发明内容
本发明制备获得了抗TNFR2的人源化抗体或其抗原结合片段，以及制备了所述抗TNFR2人源化或其抗原结合片段的药物组合物，并验证了其用途。
在第一方面，本发明提供了一种特异性结合TNFR2的人源化抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区和轻链可变区；
(1)所述重链可变区包含：
a.选自SEQ ID NO.19～27任一所示的VH序列，
b.选自与SEQ ID NO.19～27任一序列相比具有至少80％、85％、90％、91％、92％、93％、 94％、95％、96％、97％、98％、99％或100％序列一致性的VH序列，或，
c.选自与SEQ ID NO.19～27任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸插入、缺失和/或替换的VH序列；
(2)所述轻链可变区包含：
a.选自SEQ ID NO.28～37任一所示的VL序列，
b.选自与SEQ ID NO.28～37任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VL序列，或，
c.选自与SEQ ID NO.28～37任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸插入、缺失和/或替换的VL序列；
可选地，所述氨基酸插入、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
可选地，所述替换为保守氨基酸的替换。
在某些实施方案中，在本发明所述的抗体或其抗原结合片段中，所述重链可变区包含与SEQ ID NO.19～27任一序列相比CDR完全一致，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH序列；所述轻链可变区包含与SEQ ID NO.28～37任一序列相比CDR完全一致，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VL序列。
在某些实施方案中，本发明所述的抗体或其抗原结合片段包含的重链可变区和轻链可变区选自如下组：
(1)具有SEQ ID NO.19所示的VH和SEQ ID NO.28所示的VL；
(2)具有SEQ ID NO.20所示的VH和SEQ ID NO.29所示的VL；
(3)具有SEQ ID NO.19所示的VH和SEQ ID NO.30所示的VL；
(4)具有SEQ ID NO.20所示的VH和SEQ ID NO.31所示的VL；
(5)具有SEQ ID NO.19所示的VH和SEQ ID NO.31所示的VL；
(6)具有SEQ ID NO.21所示的VH和SEQ ID NO.32所示的VL；
(7)具有SEQ ID NO.22所示的VH和SEQ ID NO.32所示的VL；
(8)具有SEQ ID NO.23所示的VH和SEQ ID NO.32所示的VL；
(9)具有SEQ ID NO.24所示的VH和SEQ ID NO.32所示的VL；
(10)具有SEQ ID NO.25所示的VH和SEQ ID NO.32所示的VL；
(11)具有SEQ ID NO.26所示的VH和SEQ ID NO.33所示的VL；
(12)具有SEQ ID NO.27所示的VH和SEQ ID NO.34所示的VL；
(13)具有SEQ ID NO.27所示的VH和SEQ ID NO.35所示的VL；
(14)具有SEQ ID NO.27所示的VH和SEQ ID NO.33所示的VL；
(15)具有SEQ ID NO.27所示的VH和SEQ ID NO.36所示的VL；
(16)具有SEQ ID NO.26所示的VH和SEQ ID NO.34所示的VL；
(17)具有SEQ ID NO.27所示的VH和SEQ ID NO.37所示的VL；
(18)具有与上述(1)～(17)任一序列组合相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
(19)具有与上述(1)～(17)任一项序列组合相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸插入、缺失和/或替换的VH和VL组合；或，
(20)具有与上述(1)～(17)任一项序列组合相比CDR完全一致，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
可选地，所述氨基酸插入、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
可选地，所述替换为保守氨基酸的替换。
在某些实施方案中，在本发明所述的抗体或其抗原结合片段中，所述抗体或其抗原结合片段包含：CDR1-VH选自SEQ ID NO.1、7、或13；CDR2-VH选自SEQ ID NO.2、8、或14；CDR3-VH选自SEQ ID NO.3、9、或15；CDR1-VL选自SEQ ID NO.4、10、或16；CDR2-VL选自SEQ ID NO.5、11、或17；和CDR3-VL选自SEQ ID NO.6、12、或18。
在某些实施方案中，本发明所述的抗体或其抗原结合片段，其与人TNFR2结合的解离常数(KD)不大于7nM，与食蟹猴TNFR2结合的解离常数(KD)不大于5nM。
在某些实施方案中，本发明所述的抗体或其抗原结合片段包含或不包含重链恒定区和/或轻链恒定区；优选地，所述重链恒定区包含全长重链恒定区或其片段，所述片段可选自CH1结构域、Fc结构域或CH3结构域；优选地，所述重链恒定区和/或轻链恒定区为人重链恒定区和/或人轻链恒定区；优选地，所述重链恒定区可选自IgG重链恒定区，例如IgG1重链恒定区、IgG2重链恒定区、IgG3重链恒定区或IgG4重链恒定区；优选地，所述重链恒定区是人IgG1重链恒定区、人IgG2重链恒定区、人IgG3重链恒定区或人IgG4重链恒定区。
在某些实施方案中，本发明所述的抗体或其抗原结合片段，选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、或双抗体(diabody)。
在某些实施方案中，本发明所述的抗体或其抗原结合片段，通过或不通过接头(linker)偶联有另一分子；优选地，所述另一分子可选自治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
在某些实施方案中，本发明所述的抗体或其抗原结合片段，具有以下特性：
(1)特异性结合细胞表面表达TNFR2的细胞；(2)特异性结合Treg细胞；(3)抑制TNFα与TNFR2蛋白的结合；(4)抑制TNFα与细胞表面表达的TNFR2的结合；(5)抑制TNFα介导的Treg的增殖和/或Treg功能；(6)抑制TNFα介导的IκBα降解；(7)抑制Treg对Tcon细胞增殖的抑制；(8)介导针对TNFR2表达细胞的ADCC功能；(9)提升肿瘤浸润免疫细胞TIL中CD8+T/Treg细胞比例；或/和(10)抑制肿瘤生长。
在第二方面，本发明提供了一种多特异性抗原结合分子，所述多特异性抗原结合分子至 少包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含本发明所述的抗体或其抗原结合片段，所述第二抗原结合模块特异性结合TNFR2以外的其他抗原或结合与第一抗原结合模块不同的TNFR2抗原表位；优选地，所述其他抗原选自CD3、CD4、CD5、CD8、CD14、CD15、CD16、CD16A、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD70、CD74、CD80、CD86、CD126、CD138、B7、PD-1、PD-L1、PD-2、CTLA4、PRVIG、TIGHT、HAS、CLDN18.2、MSLN、MUC、Ia、HLA-DR、腱生蛋白、EGFR、VEGF、P1GF、ED-B纤连蛋白、癌基因产物、IL-2、IL-6、IL-15、IL-21、TRAIL-R1或TRAIL-R2；优选地，所述多特异性抗体为“双特异性”、“三特异性”或“四特异性”。
在第三方面，本发明提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述细胞外抗原结合结构域包含本发明第一方面所述TNFR2人源化抗体或其抗原结合片段，或包含本发明第二方面所述多特异性抗原结合分子。
在第四方面，本发明提供了一种免疫效应细胞，所述免疫效应细胞包含本发明第三方面所述嵌合抗原受体或包含编码本发明第三方面所述嵌合抗原受体的核酸的片段；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
在第五方面，本发明提供了一种分离的核酸片段，所述核酸片段编码本发明第一方面所述TNFR2人源化抗体或其抗原结合片段、第二方面所述多特异性抗原结合分子或第三方面所述的嵌合抗原受体。
在第六方面，本发明提供了一种载体(vector)，所述载体包含第五方面所述核酸片段。
在第七方面，本发明提供了一种宿主细胞，所述宿主细胞包含第六方面所述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)；优选地，所述细胞缺乏岩藻糖基转移酶，更优选地，所述岩藻糖基转移酶是FUT8。
在第八方面，本发明提供了一种制备本发明任一项所述抗体或其抗原结合片段或本发明所述多特异性抗原结合分子的方法，所述方法包括培养第七方面所述细胞，以及分离所述细胞表达的抗体或其抗原结合片段或多特异性抗原结合分子。
在第九方面，本发明提供了一种制备免疫效应细胞的方法，所述方法包括将编码前述CAR的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达所述CAR。
在第十方面，本发明提供了一种药物组合物，所述组合物包含本发明所述的抗体或其抗原结合片段、本发明所述的多特异性抗原结合分子、本发明所述的嵌合抗原受体、本发明所述的免疫效应细胞、本发明所述的核酸片段、本发明所述的载体、本发明所述的细胞或本发明所述方法制备获得的产品；优选地，所述组合物还包含药学上可接受的载体(carrier)、稀 释剂或助剂；优选地，所述组合物还包含额外的抗肿瘤剂、免疫治疗剂或免疫抑制剂；优选地，所述额外的抗肿瘤剂选自PD-1抗体、PD-L1抗体或CTLA-4抗体。
在第十一方面，本发明提供了所述的抗体或其抗原结合片段、所述的多特异性抗原结合分子、所述的嵌合抗原受体、所述的免疫效应细胞、所述的核酸片段、所述的载体、所述的细胞、所述方法制备获得的产品以及所述药物组合物在制备治疗和/或预防免疫异常相关疾病的药物中的用途；优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；优选地，所述疾病是癌症或自身免疫性疾病；优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力、IgG4相关性疾病。
在第十二方面，本发明提供了一种治疗和/或预防免疫异常相关疾病的方法，所述方法包括向受试者施用有效量的本发明所述的抗体或其抗原结合片段、所述的多特异性抗原结合分子、所述的嵌合抗原受体、所述的免疫效应细胞、所述的核酸片段、所述的载体、所述的细胞、所述方法制备获得的产品或所述药物组合物；优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；优选地，所述疾病是癌症或自身免疫性疾病；优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力、IgG4相关性疾病。
在第十三方面，本发明还提供了向所述对象施用额外的抗肿瘤治疗，例如化疗剂、靶向治疗剂和免疫治疗剂，包括PD-1/PD-L1治疗如抗PD-1/PD-L1抗体、抗CTLA-4治疗剂如抗CTLA-4抗体；优选地，所述额外的抗肿瘤治疗选自PD-1/PD-L1治疗；优选地，所述PD-1/PD-L1治疗选自PD-L1抗体。
在第十四方面，本发明提供了一种体外检测TNFR2的方法，其包括使怀疑含有TNFR2的样品与本发明所述的抗体或其抗原结合片段相接触的步骤。
术语定义和说明
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
如本文所用，术语“TNFR2”是指肿瘤坏死因子受体2，也称为肿瘤坏死因子受体超家族成员1B(TNFRSF1B)或CD120b，这是一种结合肿瘤坏死因子-α(TNFα)的膜受体。所述TNFR2优选地是人TNFR2。
如本文所用，术语“抗-肿瘤坏死因子受体2抗体”、“肿瘤坏死因子受体2抗体”、“抗TNFR2抗体”、“TNFR2抗体”、“抗-TNFR2抗体部分”和/或“抗-TNFR2抗体片段”等是指任何包含能够特异性结合TNFR2的免疫球蛋白分子的至少一部分(例如但不限于重链或轻链的至少一个互补决定区(CDR)或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分)的含蛋白质或肽的分子。TNFR2抗体还包括抗体样蛋白支架(如第十纤连蛋白 III型结构域(10Fn3))，其含有与抗体CDR在结构和溶剂可及性上相似的BC、DE和FG结构环。10Fn3结构域的三级结构类似于IgG重链可变区的三级结构，并且通过将10Fn3的BC、DE和FG环的残基用来自TNFR2单克隆抗体的CDR-H1、CDR-H2或CDR-H3区的残基替换，本领域技术人员可以将例如TNFR2单克隆抗体的CDR接枝到纤连蛋白支架上。
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“抗原结合片段”和“抗体片段”可互换，是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature 341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的可高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的可高变区之外。这些位置中的一个或更多个也可以在延伸的可高变区中找到。本发明包括在这些杂合可高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区， 其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
如本文所用，术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据EUKabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi) 软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
(1)酸性氨基酸：Asp(D)和Glu(E)；
(2)碱性氨基酸：Lys(K)、Arg(R)和His(H)；
(3)亲水性不带电荷氨基酸：Ser(S)、Thr(T)、Asn(N)和Gln(Q)；
(4)脂肪族不带电荷氨基酸：Gly(G)、Ala(A)、Val(V)、Leu(L)和Ile(I)；
(5)非极性不带电荷的氨基酸：Cys(C)、Met(M)和Pro(P)；
(6)芳香族氨基酸：Phe(F)、Tyr(Y)和Trp(W)。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow & Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow & Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
如本文所用，术语“多特异性抗体”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对TNFR2的抗原表位而被检测，另一个可以针对TNFR2的另一个抗原表位或任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
如本文所用，术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、 两个结合位点、四个结合位点和六个结合位点的存在。
如本文所用，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
如本文所用，术语“嵌合抗体”是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。参见例如，Morrison,1985,Science 229(4719):1202-7；Oi等人，1986,Bio Techniques 4:214-221；Gillies等人，1985J Immunol Methods 125:191-202；以上通过援引加入并入本文。
如本文所用，术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。所述另一个分子可以是治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。“抗体缀合物”例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
本文术语“抗原嵌合受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工免疫效应细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)锚定CAR进入免疫效应细胞的跨膜结构域，和(3)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
如本文所用，术语“调节性T细胞”或“Treg”，也曾被称为抑制T细胞(suppressor T cells)，是一群负调节机体免疫反应的淋巴细胞，用以维持对自身抗原的耐受性，控制免疫反应过度，避免对正常细胞产生免疫损伤，防止自身免疫病的发生。Treg表达以下生物标志物：CD4、FOXP3和CD25，被认为与幼稚型CD47细胞源自同一谱系。Treg在肿瘤的发生中起到极其重要的作用，多项研究表明Treg细胞在肿瘤微环境中的数目显著增加，包括黑色素癌，卵巢癌，乳腺癌，结肠癌，肺癌，胰腺癌等，同时Treg细胞的数目和肿瘤病人的存活率亦密切相关。此外，肿瘤细胞会诱导肿瘤浸润性Treg细胞的增殖，增殖的Treg细胞会大量分泌TGF-β等免疫抑制因子，抑制CD8+T细胞等免疫细胞的功能，阻碍免疫细胞对肿瘤的杀伤作用，是多种实体瘤和血液瘤免疫治疗失败的重要耐药机制。近期研究表明，PD-1/PD-L1等免疫治疗病人的免疫耐受也与Treg密切相关。
如本文所用，术语“载体”(vector)包括核酸载体，例如DNA载体(如质粒)、RNA载体、病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其 增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、牛、绵羊、马和野牛等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
如本文所用，术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分的用量。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
附图说明
图1.显示受试抗体和人TNFR2重组蛋白的结合能力。图A、B、C分别为#001、#219、#224受试抗体人源化分子与人TNFR2重组蛋白的结合能力；其中WT为人鼠嵌合野生型分子，抗鸡蛋白溶菌酶(anti-Hel-WT)同型对照抗体为该实验的阴性对照。
图2.显示受试抗体和食蟹猴TNFR2重组蛋白的结合能力。图A、B、C分别为#001、#219、#224受试抗体人源化分子与食蟹猴TNFR2重组蛋白的结合能力；其中WT为人鼠嵌合野生型分子，抗鸡蛋白溶菌酶(anti-Hel-WT)同型对照抗体为该实验的阴性对照。
图3.显示受试抗体和高表达人TNFR2的CHO-TNFR2细胞的结合能力。图A、B、C分别为#001、#219、#224受试抗体人源化分子与CHO-TNFR2细胞的结合能力；其中anti-Hel-WT为阴性同型对照抗体。
图4.显示受试抗体和Treg细胞的结合能力。图A、B分别为#001、#219受试抗体人源化分子与Treg结合的重叠图。空心峰为anti-Hel-WT同型阴性对照抗体的结合图，实心峰为每个抗体与Treg细胞的结合图；“只加二抗”和“未染色”(未加任何抗体的空白细胞对照) 都为阴性对照，抗体浓度为200ng/ml。
图5.显示受试抗体和激活后的食蟹猴PBMC中CD8+T细胞的结合。图A为受试抗体分别在2μg/ml与0.2μg/ml浓度与食蟹猴CD8+T结合的重叠图。图B为受试抗体分别在2μg/ml与0.2μg/ml浓度与食蟹猴CD8+T结合的平均荧光强度倍数(受试样品结合的平均荧光强度与只加二抗样品的平均荧光强度的比值)。其中抗鸡蛋白溶菌酶(anti-Hel)同型对照抗体为该实验的阴性对照。
图6.显示受试抗体抑制人TNFα与人TNFR2重组蛋白间的相互作用。图A、B、C分别为#001、#219、#224受试抗体人源化分子对TNFα-TNFR2的抑制；其中抗鸡蛋白溶菌酶(anti-Hel-WT)同型对照抗体为该实验的阴性对照。
图7.显示受试抗体抑制人TNFα与CHO-TNFR2细胞表达的人TNFR2相互作用。图A、B、C分别为#001、#219、#224受试抗体人源化分子对TNFα与CHO-TNFR2相互作用的抑制；其中anti-Hel-WT为阴性同型对照抗体。
图8.显示受试抗体对TNFα引起的IκBα降解的抑制作用。图A为本实验的圈门策略。图B为受试抗体对TNFα引起的IκBα降解的抑制作用；左边图为不同浓度的抗体与IκBα low的百分比做的柱状图，右边图为抗体浓度梯度对IκBα low的百分比相对于阴性对照抗体anti-Hel的倍数的变化。抗体的试验浓度分别在0.25，2.5和25μg/ml；其中“不做处理的Treg细胞”是指没有任何处理的Treg细胞，TNF是指用10ng/ml TNFα处理过Treg细胞；Rat IgG2b-PE为Anti-IκBα-PE抗体的阴性同型对照(anti-Hel)。每个浓度受试抗体与阴性同型对照anti-Hel相比具有统计学差异(**p<0.01,***p<0.001,Two-Way ANOVA Analysis)。
图9.显示受试抗体对TNFα诱导的Treg细胞增殖的抑制。图A为不同受试抗体与阴性同型对照anti-Hel对Treg增殖影响的重叠图，其中虚线为anti-Hel，实线为受试抗体。图B为不同抗体对TNFα诱导Treg增殖的抑制作用，纵坐标为Treg增殖百分比，横坐标为受试抗体。其中“未加药处理”(即不加任何药物处理)和anti-Hel为本实验体系的阴性对照。每个抗体的实验浓度为12.5μg/ml。每个受试抗体与阴性同型对照anti-Hel相比具有统计学差异(**p<0.01,***p<0.001,One-Way ANOVA Analysis)。
图10.显示受试抗体对Treg对Tcon细胞增殖抑制活性的抑制作用。图A为不同受试抗体与阴性同型对照anti-Hel对Treg抑制Tcon细胞增殖影响的重叠图，其中虚线为anti-Hel，实线为受试抗体。图B为不同抗体对Treg抑制Tcon细胞增殖的抑制作用，纵坐标为Tcon细胞增殖百分比，横坐标为受试抗体。其中未加药处理(即No treat)和Anti-Hel为本实验体系的阴性对照，Tcon+beads为没有Treg细胞存在时的Tcon增殖，为实验体系的阳性对照。每个抗体的实验浓度为12.5μg/ml。每个受试抗体与阴性同型对照anti-Hel相比具有统计学差异(*p<0.05,**p<0.01,One-Way ANOVA Analysis)。
图11.显示受试抗体的ADCC杀伤作用。图A示受试抗体对Treg的ADCC杀伤作用。图B示受试抗体对高表达人TNFR2的CHO-TNFR2细胞的ADCC杀伤作用。其中anti-Hel为阴性对照抗体。
图12A-12C.显示hTNFR2转基因小鼠(TNFR2 HuGEMM)血液免疫细胞中hTNFR2和mTNFR2的表达鉴定。图12A示TNFR2 HuGEMM(亦可称为hTNFR2-GEMM)小鼠血液中 免疫细胞的FACS圈门策略及圈出的CD4+T细胞，CD8+T细胞和Treg细胞上mTNFR2和hTNFR2的表达水平及圈门的FMO对照(Fluorescence Minus One control)。图12B示TNFR2 HuGEMM和Balb/c野生型小鼠血液中CD4+T细胞，CD8+T细胞及Treg细胞中mTNFR2和hTNFR2的表达水平的峰图；点线:同型,虚线:BALB/c小鼠,实线:TNFR2 HuGEMM。图12C为TNFR2 HuGEMM和Balb/c野生型小鼠血液中CD4+T细胞，CD8+T细胞及Treg细胞中mTNFR2和hTNFR2的平均荧光强度统计图。
图13A-13C.显示TNFR2抗体抑制mTNFα诱导的TNFR2 HuGEMM(图中简称GEMM)小鼠的Treg细胞增殖。图13A示GEMM小鼠和野生型WT Balb/c小鼠脾脏中Treg细胞表达人TNFR2和小鼠TNFR2的水平。图13B和13C为受试TNFR2抗体在无激活剂(B)或有激活剂(C)Dynabeads mouse T-activator CD3/CD28下对mTNFα诱导的GEMM小鼠或WT Balb/c小鼠的Treg细胞增殖的抑制作用。每个抗体的实验浓度为10μg/ml。每个受试抗体与阴性同型对照相比具有统计学差异(***p<0.001,****p<0.0001，One-Way ANOVA Analysis)。
图14.显示抗TNFR2抗体能剂量依赖性抑制TNFR2 HuGEMM小鼠肿瘤生长。图A、B分别示219-Hu1-mIgG2a、001-Hu3-mIgG2a在剂量为30mg/kg,10mg/kg,3mg/kg时相比较载剂(Vehicle)对照组可剂量依赖性抑制CT26-hTNFR2 KI肿瘤的生长。
图15.显示抗TNFR2抗体抑制TNFR2 HuGEMM小鼠肿瘤生长的同时不引起小鼠的体重下降。图A-B示不同剂量219-Hu1-mIgG2a或001-Hu3-mIgG2a给药后小鼠体重绝对重量的变化。图C-D示219-Hu1-mIgG2a或001-Hu3-mIgG2a不同剂量给药后小鼠体重的变化率。
图16.显示抗TNFR2抗体在人PBMC免疫重建人源化小鼠结肠癌肿瘤模型中的两个不同PBMC供体中都能显著抑制肿瘤生长。图A示在donor#193中，抗TNFR2抗体219-Hu1具有显著的药效。图B示在donor#272中，肿瘤接种后第29天，与对照抗体组相比，001-Hu3、219-Hu1、Keytruda组的肿瘤增长均被显著抑制，且具有统计学差异(****:p<0.0001,vs.isotype,Two way ANOVA)。图C-D示在donor#193和donor#272中，对照组和给药组小鼠均出现体重下降。
图17.显示抗TNFR2抗体能显著提升肿瘤浸润免疫细胞TIL中CD8+T/Treg细胞比例。图A示肿瘤浸润TIL中CD4+T，CD8+T及Treg细胞的圈门策略。图B示在donor#193中，isotype抗体，001-Hu3抗体及219-Hu1抗体治疗后肿瘤浸润白细胞(TIL)中CD4+T，CD8+T及Treg占CD45+细胞的比例。图C示在donor#193中，isotype抗体，001-Hu3抗体及219-Hu1抗体治疗后TIL中CD4+T，CD8+T及Treg在每mg肿瘤组织中的绝对数量。图D示在donor#193中，isotype抗体，001-Hu3抗体及219-Hu1抗体治疗后TIL中CD8+T/Treg的比例。图E示在donor#272中，isotype抗体，001-Hu3抗体及219-Hu1抗体治疗后TIL中CD4+T，CD8+T及Treg占CD45+细胞的比例。图17F示在donor#272中，isotype抗体，001-Hu3抗体及219-Hu1抗体治疗后TIL中CD4+T，CD8+T及Treg在每g肿瘤组织中的绝对数量。图17G示在donor#272中，isotype抗体，001-Hu3抗体及219-Hu1抗体治疗后肿瘤浸润TIL中CD8+T/Treg的比例(*:p<0.05,**:p<0.01,***:p<0.001,unpaired T test)。
图18.显示hTNFR2 KI-CT26.1C03结肠癌模型中各治疗组肿瘤体积变化曲线。
图19.显示hTNFR2 KI-CT26.1C03结肠癌模型中各治疗组动物体重变化曲线。本图表示 不同剂量219-Hu1-hIgG1抗体或联合anti-mPD-L1抗体给药后小鼠体重绝对重量的变化。
图20.显示hTNFR2 KI-CT26.1C03结肠癌模型中各治疗组动物体重变化率曲线。本图表示不同剂量219-Hu1-hIgG1抗体或联合anti-mPD-L1抗体给药后小鼠体重的变化率。
图21.显示hTNFR2 KI-CT26.1C03结肠癌模型中各治疗组实验终点肿瘤体重变化。本图表示不同剂量219-Hu1-hIgG1抗体或联合anti-mPD-L1抗体，在实验终点分离肿瘤并称重的肿瘤重量的变化。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1 抗TNFR2人源化单克隆抗体的表达纯化
1.1抗TNFR2抗体人源化
采用“CDRs移植”方法进行抗体人源化，即基于序列挑选同源性最高的人源性抗体提供抗体骨架区(FRs)，把目标抗体中基于Kabat命名方法的抗原结合片段互补决定区(CDRs)，移植到前者形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的人源化抗体可变区序列。其次，为有效保持抗体活性和亲和力，基于抗体结构建模(MOE软件)，选择潜在回复突变点，选择标准：1).选择抗体骨架区位于VH-VL界面、靠近或与CDRs有直接相互作用等氨基酸残基进行回复突变，这类氨基酸残基对保持CDRs区构象多较重要；2).考虑到免疫原性，尽量选择包埋在蛋白内部的氨基酸进行回复突变；3).考虑到抗体稳定性和表达水平，优选分子能量降低的突变。通过测试含有不同突变的人源化抗体与人TNFR2的亲和力以及和表面表达TNFR2的细胞的结合，筛选与鼠源抗体亲和力、抗体表征和活性功能相当或更好的人源化抗体。
其中，TNFR2抗体Kabat命名方法的抗原结合片段互补决定区(CDRs)详见表1，表2示出人源化抗体分子的VH和VL序列，表3示出人源化抗TNFR2抗体的VH和VL序列配对情况。
表1.候选抗体CDRs的KABAT分析结果(人源化抗体CDR同鼠抗体CDR)
Figure PCTCN2022074228-appb-000001
Figure PCTCN2022074228-appb-000002
表2.人源化抗TNFR2抗体的VH和VL序列
Figure PCTCN2022074228-appb-000003
Figure PCTCN2022074228-appb-000004
表3.人源化抗TNFR2抗体的VH和VL序列配对情况
抗体ID VH VL
001-Hu1 VH1(SEQ ID NO.19) VL1(SEQ ID NO.28)
001-Hu2 VH2(SEQ ID NO.20) VL2(SEQ ID NO.29)
001-Hu3 VH1(SEQ ID NO.19) VL3(SEQ ID NO.30)
001-Hu4 VH2(SEQ ID NO.20) VL4(SEQ ID NO.31)
001-Hu5 VH1(SEQ ID NO.19) VL4(SEQ ID NO.31)
219-Hu1 VH3(SEQ ID NO.21) VL5(SEQ ID NO.32)
219-Hu2 VH4(SEQ ID NO.22) VL5(SEQ ID NO.32)
219-Hu3 VH5(SEQ ID NO.23) VL5(SEQ ID NO.32)
219-Hu4 VH6(SEQ ID NO.24) VL5(SEQ ID NO.32)
219-Hu5 VH7(SEQ ID NO.25) VL5(SEQ ID NO.32)
224-Hu1 VH8(SEQ ID NO.26) VL6(SEQ ID NO.33)
224-Hu2 VH9(SEQ ID NO.27) VL7(SEQ ID NO.34)
224-Hu3 VH9(SEQ ID NO.27) VL8(SEQ ID NO.35)
224-Hu4 VH9(SEQ ID NO.27) VL6(SEQ ID NO.33)
224-Hu5 VH9(SEQ ID NO.27) VL9(SEQ ID NO.36)
224-Hu6 VH8(SEQ ID NO.26) VL7(SEQ ID NO.34)
224-Hu7 VH9(SEQ ID NO.27) VL10(SEQ ID NO.37)
1.2抗TNFR2人源化抗体的表达
转染前一天，将ExpiCHO-S细胞进行计数，并以2.5～4×10 6细胞/mL的密度接种于新鲜预热的ExpiCHO表达培养基(英维捷基，A291002)中过夜培养。转染当天，取过夜培养的ExpiCHO-S细胞悬液进行计数，细胞浓度大概在7～10×10 6细胞/mL，转染用的细胞活率大于95％。根据转染个数，取所需的细胞数目，用ExpiCHO表达培养基稀释至终浓度为6×10 6细胞/mL的密度，置于37℃，8％CO 2，100rpm摇床备用。转染准备：将质粒用OptiPRO TMSFM培养基(英维捷基，12309019)中稀释，轻轻摇转离心管使其混匀。将混匀的ExpiFectamine TMCHO试剂(英维捷基，A29129)加入到质粒稀释液中，轻轻摇转离心管使其混匀后室温静置2分钟。将上述的质粒/ExpiFectamine TMCHO试剂复合物缓慢滴入待转染的细胞悬液中，在滴加的过程中摇转摇瓶。转染结束后，将转染的细胞放入温度37℃，8％CO 2，100rpm的摇床中培养。转染后第一天，向转染的细胞中补加0.6％ExpiFectamine TMCHO Enhancer(英维捷基，A29129)和16％ExpiCHO TMFeed(英维捷基，A29129)，加入过程中轻轻摇转摇瓶，并且将细胞转移至温度32℃，5％CO 2，100rpm的摇床中培养。转染后第五天，向转染的细胞中补加16％ExpiCHO TMFeed，加入过程中轻轻摇转摇瓶。转染后第十二天，收集细胞上清，并于4000g离心10分钟吸取上清，并进一步纯化抗体。
1.3抗TNFR2人源化抗体的纯化
经高速离心收集的细胞培养液上清，用0.45+0.22μM滤膜进行过滤，利用Akta Avant 150(Cytiva)进行亲和层析进行第一步纯化。层析介质为与Fc相互作用的Protein A填料Mabselect PrismA(Cytiva，货号：17549803)，平衡缓冲液为PBS(2.5g/L Na 2HPO 4 12H 2O，0.408g/L NaH 2PO 4，8.76g/L NaCl，pH7.2)，平衡4倍柱体积后，将细胞上清上样结合，流速控制为样品在柱上保留时间≥5min。上样结束后，用PBS(pH 7.2)冲洗柱子，直至A280紫外吸收降至基线。然后用20mM PB+1M NaCl(3.752g/L Na 2HPO4·12H 2O，1.314g/L NaH 2PO 4，58.44g/L NaCl pH6.2)淋洗2倍柱体积。再用PBS(pH 7.2)冲洗柱子，直至A280紫外吸收和电导达到基线。最后用20mM柠檬酸(2.184g/L柠檬酸，1.086g/L柠檬酸钠，pH 3.4)的洗脱缓冲液冲洗层析柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样品用1M Tris(121.14g/L Tris)中和并用pH计Seven2Go(梅特勒-托利多)检测至中性。收集的抗体经SEC-HPLC鉴定纯度大于95％并用于后续研究。
实施例2 ELISA检测TNFR2人源化抗体与人和食蟹猴TNFR2蛋白的特异性结合
酶标板中预先包被100μl/孔的0.5μg/ml人TNFR2(Human TNFR2-His,Sino 10417-H08H)或食蟹猴TNFR2(Cynomolgus TNFR2-His,Sino 90102-C08H)；将受试抗TNFR2人源化抗体(实施例1所述可变区连接IgG-Fc构成；WT嵌合抗体同样采用IgG-Fc结构)进行3.33倍梯度稀释，100μl/孔加样，室温振荡孵育1.5小时；洗板后加入鼠抗人(mouse anti-human)IgG Fc-HRP工作液(1：10000稀释)，100μl/孔加样，室温振荡孵育1.0小时；再次洗板，加入HRP的底物TMB进行显色，加入终止液终止反应后用酶标仪(MD I3)读取吸光值。以抗体浓度为横坐标，对应的OD值为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值。EC50值越小，抗体与人/食蟹猴TNFR2结合的能力越强。将人源化抗体的结合效果均归一化到与其对应的人鼠嵌合抗体WT(序列参见SEQ ID NO.38-43)，百分比值越高，说明人源化抗体结合的效果越好。抗TNFR2抗体与人TNFR2蛋白的结合结果见图1和表4，与食蟹猴TNFR2的结合结果见图2和表4。数据表明，所有受试抗体均能和人或猴TNFR2蛋白特异性结合，且人源化抗体与人鼠嵌合抗体的结合能力基本一致。
表4.抗TNFR2抗体与人或食蟹猴TNFR2蛋白特异性结合的ELISA结果
Figure PCTCN2022074228-appb-000005
Figure PCTCN2022074228-appb-000006
001-WT VH氨基酸序列(SEQ ID NO.38)：
Figure PCTCN2022074228-appb-000007
001-WT VL氨基酸序列(SEQ ID NO.39)：
Figure PCTCN2022074228-appb-000008
219-WT VH氨基酸序列(SEQ ID NO.40)：
Figure PCTCN2022074228-appb-000009
219-WT VL氨基酸序列(SEQ ID NO.41)：
Figure PCTCN2022074228-appb-000010
224-WT VH氨基酸序列(SEQ ID NO.42)：
Figure PCTCN2022074228-appb-000011
224-WT VL氨基酸序列(SEQ ID NO.43)：
Figure PCTCN2022074228-appb-000012
实施例3 BIAcore检测TNFR2人源化抗体与人和食蟹猴TNFR2蛋白的亲和力
利用Biacore检测受试抗TNFR2抗体与人和食蟹猴TNFR2蛋白之间的特异性结合。该实验采用Protein A芯片，通过手工操作(manual run)测定出芯片捕获稀释后的抗体所需要的时间，以使得能饱和结合抗原Rmax为50RU。将人(Human TNFR2-His,Sino 10417-H08H)和食蟹猴(Cynomolgus TNFR2-His,Sino 90102-C08H)TNFR2均梯度稀释至32,16,8,4,2nM。采用多循环动力学测得抗体与抗原的亲和力。在每一个循环中，注射抗体后再注入梯度浓度的TNFR2蛋白，使抗原与抗体发生结合与解离过程。每个循环后用Glycine pH1.5进行Protein A芯片的再生(去除芯片上的蛋白)。应用BIAcore T200分析软件拟合抗体抗原的亲和力KD。表5结果可知，所有受试抗TNFR2抗体与人或食蟹猴TNFR2蛋白之间存在特异性结合，且亲和力水平较高。
表5.抗TNFR2抗体与人或食蟹猴TNFR2蛋白特异性结合的BIAcore结果
Figure PCTCN2022074228-appb-000013
Figure PCTCN2022074228-appb-000014
实施例4 FACS检测TNFR2抗体与CHO-TNFR2细胞表面人TNFR2的结合
取转染了人TNFR2高表达质粒的CHO-K1稳定细胞(命名为CHO-TNFR2)，转染的人TNFR2全长质粒购自Sino Biological(货号:HG10417-UT，NCBI Ref Seq:NM_001066.2),在细胞密度不超过80％时进行实验。弃去细胞培养基，用PBS润洗并加入1ml胰酶消化2分钟，用含10％FBS的Ham’s F12完全培养基终止消化后制成细胞悬液。计数后，取适量细胞悬液，350×g离心，弃上清，按照1×10 7个细胞/ml的密度，加入相应体积的封闭液(10％FBS+PBS)将细胞重悬，4℃孵育30分钟。孵育结束后，350×g离心去上清，用染色缓冲液(2％FBS+PBS)将细胞重悬为2×10 6个细胞/ml的密度，铺入96孔板，每孔加入50μl的细胞悬液，待用。用PBS将抗体从最高浓度20μg/ml(两倍浓度)开始进行10倍梯度稀释，将稀释好的抗体加至已含有50μl细胞悬液的孔中，置于微孔板振荡器上500rpm震荡1分钟，使抗体与细胞充分混合，4℃孵育1h。孵育结束后用染色缓冲液清洗细胞两次，每孔100μl，350×g离心5分钟弃上清。用染色缓冲液将PE goat anti-Human IgG Fc抗体(ebioscience，货号：12-4998-82)稀释250倍，以每孔100μl的体积加至洗完的细胞孔中，混合均匀，4℃染色30分钟。染色结束后同样用染色缓冲液清洗两次，最后用200μl染色缓冲液重悬细胞，流式上机检测信号(Thermo Attune NxT)，信号越强，表示抗体与TNFR2的结合能力越强。以抗体浓度为横坐标，对应的平均荧光强度MFI倍数为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值。EC50值越小，抗体与CHO-TNFR2细胞结合的能力越强。将人源化抗体的结合效果均归一化到与其对应的人鼠嵌合WT抗体，百分比值越高，说明人源化抗体结合的效果越好。如图3和表6所示检测所有受试抗TNFR2抗体均可与CHO-TNFR2细胞表面人TNFR2结合，且人源化抗体与人鼠嵌合WT抗体的结合能力基本一致。
表6.抗TNFR2抗体与CHO-TNFR2细胞表面人TNFR2特异性结合的流式结果
Figure PCTCN2022074228-appb-000015
Figure PCTCN2022074228-appb-000016
实施例5 FACS检测抗体与人调节性T细胞(Treg细胞)表面TNFR2的结合实验
人Treg细胞使用分选试剂盒(Stemcell,货号货号:18063)从人PBMC中分离获得，经过Dynabeads Human Treg Expander(Gibco,货号:11129D)体外刺激扩增15天后获得并分装冻存。取体外分离扩增的Treg细胞复苏过夜，第二天300×g离心5分钟，用DPBS重悬得到细胞悬液，计数。取实验所需的细胞数加入离心管，300×g离心5min，去上清，用染色缓冲液将细胞密度调整为2×10 6/ml，铺96孔板，每孔50μl。取所有受试抗体与对照抗体anti-Hel isotype(用PBS分别稀释到200ng/ml，总量为100μl)。将各个样品加到每个孔中，每孔50μl。将加有细胞悬液与抗体的孔板置于微孔板振荡器上，以500rpm的速度振荡1min，将细胞与抗体充分混匀。混匀后，将孔板置于4℃冰箱，孵育60min。孵育结束后，每孔加入100μl染色缓冲液，350×g离心5min，弃上清；每孔再加入200μl染色缓冲液重悬细胞，350×g离心5min，弃上清。将染色缓冲液按照250:1(染色缓冲液：荧光染色抗体)的比例加入PE goat anti-Human IgG Fc，制成染液，混匀后加入细胞孔，每孔100μl；将孔板置于微孔板振荡器上以500rpm的速度振荡1min，以将细胞与染液充分混匀。混匀后置于4℃冰箱，孵育30min。将细胞清洗两遍，最后每孔加入200μl的PBS将细胞重悬，流式上机检测信号(Thermo Attune NxT)。图4显示所有抗TNFR2人源化抗体均能与人Treg细胞有效结合。
实施例6 FACS检测抗体与激活后食蟹猴PBMC细胞的结合实验
冻存的食蟹猴PMBC细胞复苏后，用完全培养基(RPMI1640-Glutamax+10％FBS+1×P/S+1×ITS+50μMPM巯基乙醇)重悬得到细胞悬液，计数。根据计数结果重悬细胞密度为5×10 6/ml，在96孔U底板中，每孔铺100μl的细胞悬液。配置两成浓度的T细胞激活试剂Immunocult T cell(Stemcell,1：50)activator+rhIL-2(R&D,200IU)，将两成浓度的激活试剂加入到细胞中，每孔100μl，混匀后放置37℃，5％CO 2培养箱中培养三天。收 集激活后的细胞进行计数，染色缓冲液(含2％FBS的DPBS溶液)将细胞密度调整为4×10 6/ml，铺96孔V底板，每孔50μl。取所有受试抗体与anti-Hel对照抗体(用染色缓冲液分别稀释到0.4μg/ml和4μg/ml)，将各个样品加到每个孔中，每孔50μl。将加有细胞悬液与抗体的孔板置于微孔板振荡器上，以500rpm的速度振荡1min，将细胞与抗体充分混匀。混匀后，将孔板置于4℃冰箱，孵育30min。孵育结束后，每孔加入100μl染色缓冲液，350×g离心5min，弃上清；每孔再加入200μl染色缓冲液重悬细胞，350×g离心5min，弃上清。加入抗体Anti-Human CD8-FTIC(BD-555366，1μl/test)和PE goat anti-Human IgG Fc(Invitrogen-124998-82,0.5μl/test)；将孔板置于微孔板振荡器上以500rpm的速度振荡1min，以将细胞与染液充分混匀。混匀后置于4℃冰箱，孵育30min。将细胞清洗两遍，最后每孔加入200μl的染色缓冲液将细胞重悬，流式上机检测信号(Thermo Attune NxT)。图5显示Anti-Hel阴性同型对照抗体不与食蟹猴CD8T细胞结合，所有受试抗TNFR2人源化抗体均能与激活后的食蟹猴PBMC中的CD8+T细胞有效结合。
实施例7 ELISA检测抗TNFR2抗体阻断TNFα与TNFR2的结合
酶标板中预先包被1μg/ml 100μl/孔的人TNFR2(Novoprotein,货号C830)，将所有受试TNFR2抗体从最高浓度30μg/ml进行2.5倍梯度稀释，稀释后的抗体分别与15ng/ml人TNFα-Biotin(Acro Biosystem,货号TNA-H82E3)等体积混合，按100μl/孔加入酶标板中，室温振荡孵育2.0小时；洗板后加入Streptavidin-HRP工作液(1:10000稀释)，100μl/孔，室温振荡孵育40分钟；再次洗板，加入HRP的底物TMB进行显色，加入终止液终止反应后用酶标仪(MD I3)读取吸光值。以抗体浓度为横坐标，对应的OD值为纵坐标绘制抗体的抑制曲线，四参数拟合(GraphPad Prism9)，计算IC50值。IC50值越小，抗体抑制人TNFα与人TNFR2结合的能力越强。将人源化抗体的阻断效果均归一化到与其对应的人鼠嵌合抗体WT，百分比值越高，说明人源化抗体抑制的效果越好。受试抗体的阻断曲线见图6，抑制活性见表7。从图6及表7可知，所有受试抗体均能显著地抑制人TNFα与人TNFR2蛋白的结合，且人源化抗体与人鼠嵌合WT抗体的抑制能力基本一致。
表7.受试抗体阻断TNFα和TNFR2的结合
Figure PCTCN2022074228-appb-000017
Figure PCTCN2022074228-appb-000018
实施例8抗TNFR2抗体阻断高表达人TNFR2的CHO细胞上TNFR2与TNFα的结合
采用高表达人TNFR2的CHO稳定细胞进行实验(同实施例4中CHO-TNFR2)。将CHO-TNFR2细胞消化后，DPBS洗涤2次，经Live/Dead(L/D)染色(室温20分钟)后，按1×10 5细胞/50μl/孔进行铺板；受试抗TNFR2抗体用染色缓冲液稀释至6μg/ml作为起始浓度，3.33倍梯度稀释，共7个浓度点；稀释后的抗体按50μl/孔加入铺好的细胞中，轻柔吹打混匀，4℃孵育30分钟。之后加入100μl/孔稀释至100ng/ml的人TNFα-biotin轻柔吹打混匀，混匀后4℃孵育30分钟。染色缓冲液洗涤2次后，加入100μl/孔的0.12μg/ml PE-streptavidin(BioLegend，货号405204)4℃孵育30分钟。染色缓冲液洗涤2次后，150μl重悬，上流式机器检测(Thermo Attune NxT)。以抗体浓度为横坐标，对应的MFI值为纵坐标绘制抗体的抑制曲线，四参数拟合(GraphPad Prism9)，计算IC50值。IC50值越小，抗体抑制人TNFα与人TNFR2结合的能力越强。将人源化抗体的阻断效果均归一化到与其对应的人鼠嵌合WT抗体，百分比值越高，说明人源化抗体抑制的效果越好。受试抗体的阻断曲线见图7，抑制活性见表8。从图7及表8可知，所有受试抗体均能显著地抑制TNFα与CHO-TNFR2细胞上TNFR2的结合。
表8.TNFR2抗体阻断TNFα和CHO细胞表面TNFR2的结合
Figure PCTCN2022074228-appb-000019
实施例9 FACS检测抗体抑制TNFα引起的人Treg细胞IκBα降解实验
人Treg细胞使用分选kit(Stemcell,货号货号:18063)从人PBMC中分离获得，经过Dynabeads Human Treg Expander(Gibco,货号:11129D)体外刺激扩增15天后获得并分装冻存。取体外分离扩增的Treg细胞复苏过夜，第二天300×g离心5分钟，用AIM-V培养基(品牌Gibco，货号31035025)，计数，根据细胞计数结果调整细胞密度至6×10 6个细胞/ml，铺96孔板，每孔25μl。取所有受试抗体与anti-Hel对照抗体(用AIM-V培养基分别稀释到四成浓度，1μg/ml，10μg/ml和100μg/ml)。将每个浓度的抗体加到对应的细胞孔中，每孔25μl。多通道移液枪混合后，放于37℃，5％CO 2培养箱中预孵育30min。使用AIM-V培养基将TNFα(品牌Novoprotein，货号C008)稀释至工作浓度的两倍即20ng/ml，向需要TNFα激活孔中加入50μl的TNFα，非激活对照孔加入50μl的AIM-V培养基，使抗体最终浓度为1乘，TNFα的最终浓度为10ng/ml，移液枪吹打混匀，将培养板放入37℃培养箱中孵育10min。孵育完成后，取出培养板，加入100μl预冷的DPBS，4℃进行离心300g，5min。然后细胞用100μl 1×live/dead violet(预冷DPBS稀释500倍)，冰上反应20min。每孔加入100μl染色缓冲液，4℃进行300g离心，5min。每孔加入100μl的1×fix-perm buffer(品牌BD，货号554714)，混合均匀，4℃固定30min。固定结束后，每孔加入100μl的1×perm buffer终止固定，400g离心5min，弃上清。再向每孔加入200μl 1×perm buffer二次洗涤细胞。向实验孔加入100μl IκBα染液(用1×perm buffer稀释100倍)，对照孔加入Rat IgG2bκ同型对照抗体1μl，混合均匀，4℃孵育30min。孵育结束后每孔加入100μl的1×perm buffer终止固定，400g离心5min，弃上清。再向每孔加入200μl 1×perm buffer二次洗涤细胞。上流式机器检测(Thermo Attune NxT)。流式检测分析活细胞中IκBα low的百分比或所有活细胞的IκBα的MFI。IκBα low的比例越低，说明抗TNFR2抗体抑制TNFα引起的IκBα降解能力越强。图8结果显示阴性对照抗体Anti-Hel对IκBα表达没有影响，而受试抗TNFR2人源化抗体均能抑制TNFα引起的IκBα降解。
实施例10 TNFR2抗体抑制TNFα和IL-2诱导的Treg细胞增殖
通过测定TNFR2抗体存在下TNFα和IL-2诱导Treg细胞的增殖情况，判断该抗体对TNFα和IL-2诱导Treg细胞增殖的抑制作用(Zaragoza B et al.,Nat Med.2016Jan；22(1):16-7)。取体外扩增分离后冻存的Treg细胞复苏过夜，第二天400×g离心5分钟，用培养基重悬得到细胞悬液，计数。将Treg用CellTrace Violet cell proliferation kit(Thermo货号C34557)进行染色，每1×10 7细胞使用1μl储存液配置成1ml染色液。37℃染色20min，用至少5倍体积的完全培养基中和反应，静置5min,离心400×g 5min。用培养基重悬清洗一次细胞，再次离心取上清。按照96孔板每孔1×10 5细胞密度进行铺板，每1×10 5细胞用50μl培养基的密度重悬细胞，转移至96孔板中。每孔加入50μl待测抗体，抗体的终浓度为12.5μg/ml，培养箱孵育30分钟。随后再每孔加入含IL-2(终浓度为300IU)和TNFα(终浓度50ng/ml)的培养基50μl，以及含有anti-CD3/CD28 Dynabeads(Gibco，货号：11129D，beads与Treg细胞比例为1：20)培养基50μl，补齐每孔终体积为200μl。每个条件设置两个平行孔，将上述孔板混匀后放入37℃培养箱培养三天进行FACS检测(Thermo Attune NxT)。通过Treg增殖的比例判断该抗体对TNFα和IL-2诱导的Treg增殖的抑制作用。结果如图9显示，相比对照anti-Hel抗体，所有抗TNFR2人源化抗体均可有效抑制TNFα和IL-2诱导的Treg细胞增殖，且具有显著性差异。
实施例11抗TNFR2抗体对Treg功能的抑制
通过测定含有抗TNFR2抗体的条件下Treg细胞对效应(responder)T细胞的抑制功能，判断抗体对Treg细胞抑制功能的抑制效果。取实验所需的Treg(CD4+CD25+FoxP3+T细胞)和Conventional T细胞(Tcon)效应细胞(CD4+CD25-T细胞)复苏过夜，第二天400×g离心5分钟，用完全培养基(RPMI1640-Glutamax+10％FBS+1×P/S+1×ITS+50μMPM巯基乙醇)重悬得到细胞悬液，计数。将Tcon细胞用CellTrace Violet cell proliferation kit(Thermo货号C34557)进行染色，每1×10 7细胞使用1μl储存液配置成1ml染色液。37℃染色20min，用至少5倍体积的完全培养基中和反应，静置5min,离心400×g 5min。用培养基重悬清洗一次细胞，再次离心弃上清。按照96孔板每孔1×10 5细胞密度进行铺板，每1×10 5细胞用50μl培养基的密度重悬细胞，转移至50mL离心管中。同时准备Treg细胞，Treg细胞和Tcon细胞按照1:1的比例进行共培养。根据Tcon细胞的数量，按照1/10的比例准备相应的anti-CD3/CD28 Dynabeads(Gibco，货号：11129D)。按照每孔50μl的体积加入Treg和Tcon的培养基中。每孔加入待测抗体，抗体的终浓度为12.5μg/ml。每个条件设置三个平行孔，将上述孔板混匀后放入37℃培养箱培养4天进行流式检测。Tcon增殖比例越高，说明抗体抑制Treg的能力越强。抑制结果如图10所示，三个受试抗TNFR2人源化抗体均能够显著抑制Treg对Tcon细胞的抑制作用，促进Tcon的增殖，且具有统计学差异。
实施例12 FACS检测抗TNFR2抗体介导的抗体依赖的细胞毒(ADCC)活性
提前一天将PBMC和冻存的Treg细胞培养于完全培养基(RPMI1640-Glutamax+10％FBS+1×P/S+1×ITS+50μMPM巯基乙醇),其中PBMC需要添加100IU/ml的IL-2。实验当天根据分离试剂盒(Stemcell，货号17955)要求将NK细胞从PBMC中分离出来，用完全培养基重悬(不含IL-2)，密度0.8×10 6/ml。用Cell Trace violet试剂标记靶细胞Treg细胞或CHO-TNFR2细胞，标记结束后将密度调整到0.4×10 6/ml。用完全培养基将待测抗体梯度稀释至终浓度的4倍，待用。按照实验板分布图将靶细胞种入细胞板中，每孔50μl,接着将稀释好的药物铺入对应的孔中，与靶细胞37℃共孵育30分钟。孵育结束后向需要加入效应细胞的孔中加入100μl的效应细胞悬液，不需要的孔补入100μl的培养基，37℃共孵育4h。反应结束后每孔加入1μl的PI染料，上机检测。靶细胞中PI阳性比例越高说明ADCC作用越显著。ADCC实验结果如图11所示，anti-Hel阴性对照抗体因没有作用靶点，没有出现ADCC杀伤作用；三个受试人源化抗体在靶细胞Treg或CHO-TNFR2细胞上均显示有ADCC活性作用。
实施例13 TNFR2转基因人源化小鼠(TNFR2 HuGEMM)hTNFR2和mTNFR2表达检测
采用CRISPR/Cas9技术,通过同源重组的方式，在Tnfrsf1b基因exon2位点定点敲入hTNFRSF1B-WPRE-PA表达框，获得Tnfrsf1b基因定点敲入hTNFRSF1B-WPRE-PA的杂合子BALB/c小鼠，然后通过繁育获得表达人Tnfrsf1b(TNFR2)基因的纯合子转基因小鼠(TNFR2 HuGEMM)。取6-8周龄雌性BALB/c小鼠(来自北京维通利华实验动物技术有限公司)和TNFR2 HuGEMM小鼠外周血，ACK(Gibco，货号A1049201)处理去除红细胞，进行FACS的免疫分型，检测小鼠外周血中CD4+T细胞、CD8+T细胞和CD4+CD25+Foxp3+ Treg细胞上mTNFR2和hTNFR2的表达情况，所有细胞群体的画门依据都为FMO对照(图12A)。结果显示TNFR2 HuGEMM小鼠不表达mTNFR2，CD4+T细胞、CD8+T细胞、Treg细胞表面hTNFR2的表达谱/表达水平与BALB/c背景小鼠上mTNFR2的表达谱/表达水平一致，表明TNFR2 HuGEMM小鼠完成了hTNFR2对mTNFR2的替代(图12B-12C)。
实施例14抗TNFR2抗体抑制TNFα诱导的TNFR2 HuGEMM的Treg细胞增殖
通过测定TNFR2抗体存在下TNFα诱导TNFR2 HuGEMM小鼠Treg细胞的增殖情况，验证TNFR2 HuGEMM小鼠经基因改造后TNFR2信号通路及相应功能得以保留，确保改造后的转基因小鼠可以用于候选抗TNFR2抗体的体内药效评价。取5只TNFR2 HuGEMM小鼠和5只野生型Balb/c小鼠，在生物安全柜中无菌取出小鼠脾脏进行研磨，将磨好的细胞悬液全部转移至15ml离心管中，离心(320g，7分钟，4℃)。倾倒上清液。加入约2-3ml ACK红细胞裂解液(Gibco，货号A1049201)，混匀，室温放置3-5分钟，直至红细胞裂解完全，加入6-8ml RPMI1640培养基终止裂解，颠倒混匀。离心(200g，8分钟，4℃)。离心完成后，倾倒上清液用EasySep buffer重悬细胞，40μm滤膜过滤细胞，进行细胞计数。然后根据Mouse CD4+CD25+Regulatory T kit(品牌Stemcell，货号18783A)说明书进行分选Treg细胞。分好的Treg细胞用CellTrace Violet cell proliferation kit(Thermo货号C34557)进行染色，每1×10 7细胞使用1μl储存液配置成1ml染色液。37℃染色20min，用至少5倍体积的完全培养基中和反应，静置5min,离心400×g 5min。用培养基重悬清洗一次细胞，再次离心取上清。按照96孔板每孔1×10 5细胞密度进行铺板，每1×10 5细胞用50μl培养基的密度重悬细胞，转移至96孔板中。每孔加入50μl待测抗体(包括三个人源化TNFR2抗体，一个抗鼠TNFR2替代抗体Surrogate antibody clone#75-54.7(BioXcell,BE0247)以及同型对照抗体anti-Hel-mIgG1)，抗体的终浓度为10μg/ml，培养箱孵育30分钟。随后再每孔加入含小鼠IL-2(品牌义翘神州，货号51061-MNAE，终浓度为300IU)以及小鼠TNFα(品牌义翘神州，货号20180502，终浓度50ng/ml)的培养基50μl，以及不含有或者含有Dynabeads mouse T-activator CD3/CD28(Gibco，货号：11452D，beads与Treg细胞比例为1：20)培养基50μl，补齐每孔终体积为200μl。同时设置Treg细胞只加小鼠IL-2，Treg细胞只加小鼠TNFα，Treg细胞加小鼠IL-2和人TNFα的对照孔。每个条件设置两个平行孔，将上述孔板混匀后放入37℃培养箱培养三天进行FACS检测(Thermo Attune NxT)。通过Treg增殖的比例判断该抗体对TNFα诱导的Treg增殖的抑制作用。图13A显示TNFR2 HuGEMM小鼠的Treg细胞表达人TNFR2，不表达鼠TNFR2；WT Balb/c小鼠的Treg细胞只表达鼠TNFR2。图13B和13C显示,无论体系中是否有Dynabeads mouse T-activator CD3/CD28激动剂，TNFR2 HuGEMM Treg在加入了mIL-2和mTNFα或hTNFα下，增殖明显增加，说明TNFR2 HuGEMM小鼠基因改造后TNFR2介导的信号得以保留。而WT balb/c只在加入了mIL-2和mTNFα下增殖增加，而hTNFα没有影响增殖，说明hTNFα不与mTNFR2结合。TNFR2 HuGEMM Treg在mIL-2和mTNFα存在下加入抗TNFR2人源化抗体，与阴性对照anti-Hel-mIgG1相比，001-Hu3-mIgG1和219-Hu1-mIgG1可显著抑制TNFR2 HuGEMM Treg的增殖，且具有统计学差异(***p<0.001)，而抗鼠TNFR2替代抗体TR75-54.7无抑制作用；对于WT Balb/c Treg在mIL-2和mTNFα存在下加入抗TNFR2抗体，与阴性对照Hamster IgG相比，抗鼠TNFR2替代抗体TR75-54.7可显著抑制WT Balb/c Treg的增殖，且具有统计学差异(***p<0.001)， 而001-Hu3-mIgG1和219-Hu1-mIgG1均无抑制作用。
实施例15抗TNFR2抗体在TNFR2 HuGEMM小鼠肿瘤模型的药效评价
采用百奥赛图基因生物技术有限公司的基于CRISPR/Cas9开发的EGE系统，将小鼠CT26细胞系中的mTNFR2基因的部分Exon2和Exon3替换为PuroR cassette，以达到基因敲除的目的，破坏小鼠内源mTNFR2的表达，然后插入小鼠胞内区、跨膜区于人胞外区嵌合蛋白的CDS，以制备只表达hTNFR2基因的CT26细胞系(CT26-hTNFR2 KI)。利用TNFR2 HuGEMM建立敲入hTNFR2基因的CT26-hTNFR2 KI细胞系结肠癌动物模型并进行抗TNFR2抗体的药效实验。
细胞库中取出一支CT26-hTNFR2 KI细胞，用完全1640培养基(1640(Gibco，61870-036)+10％FBS(Gibco，10099-141C)+1％P/S(Gibco，15140-122)+1％NEAA(Gibco，11140-050)+1％Sodium Pyruvate(Gibco，11360-070)+16μg/ml puromycin(Gibco，A11138-03))复苏细胞，复苏后的细胞置细胞培养瓶中(在细胞瓶壁标记好细胞种类、代数、日期、培养人姓名等)置于CO 2培养箱中培养(培养箱温度37℃，CO 2浓度为5％)；待细胞铺满培养瓶底部80％-90％后传代，传代后细胞继续置于CO 2培养箱中培养，重复该过程直至细胞数目满足体内药效实验的需求，随后制备细胞悬液，使用全自动细胞计数仪(BECKMAN，Vi-Cell XR)计数，根据计数结果用PBS(HyClone，SH30256.01)溶液重悬细胞，制备成细胞悬液(密度2×10 6/ml)。在小鼠的右侧皮下接种上述细胞悬液100μl/只，当肿瘤体积达到约100mm 3左右时，挑选个体肿瘤体积适中的小鼠入组，将动物按肿瘤体积使用基于EXCEL随机数的随机分组方法，分配到8个实验组，分组见表9，并于当天开始给药(标记为Day 0)，给药体积10ml/kg，溶媒PBS，腹腔注射给药，每3天给药一次，一共给药4次。每周固定时间测量肿瘤体积和体重三次。肿瘤体积计算公式：肿瘤体积(mm 3)＝长(mm)×宽(mm)×宽(mm)/2，肿瘤抑制率计算公式为：肿瘤抑制率计算公式：TGI(％)＝[1-T/C]×100％，均采用Two-way ANOVA的统计学方式，进行实验数据的分析。在给药后的第11天，和对照组相比，测试的抗TNFR2抗体组的各个剂量组和PBS对照组相比均具有统计学差异，药效明显；且相同抗体间的不同剂量组间也有剂量依赖关系，同时，10mg/kg的阳性药anti-mPD-1(BioXcell，BE0146)的药效和测试抗TNFR2抗体219-Hu1-mIgG2a和001-Hu3-mIgG2a在3mg/kg的剂量下的药效相当，以上结果说明测试的抗TNFR2人源化抗体具有显著的体内肿瘤生长抑制药效，且有剂量依赖关系(图14中A和B，表10)。同时各组实验动物在给药期间活动和进食状态良好，体重以及体重变化率均有一定程度的上升，说明测试抗TNFR2人源化抗体的体内安全性风险较低(图15中A-D)。
表9.TNFR2 HuGEMM小鼠体内药效实验分组表
Figure PCTCN2022074228-appb-000020
Figure PCTCN2022074228-appb-000021
表10.TNFR2 HuGEMM小鼠体内肿瘤抑制量效关系
Figure PCTCN2022074228-appb-000022
*:p<0.05,***:p<0.001,****:p<0.0001,Two-way ANOVA,vs.G1PBS对照组
实施例16抗TNFR2抗体在人PBMC免疫重建人源化小鼠结肠癌肿瘤模型中的药效评价
选用7-9周NOG雌性小鼠(购自北京维通利华实验动物技术有限公司)皮下接种3.5×10 6HT-29细胞(设为实验第0天)，当天腹腔注射7×10 6人外周血单核细胞(PBMC,donor#193和donor#272,由中美冠科生物技术(太仓)有限公司依据SOP-CP-042从已筛选出捐献者全血中提取)，建立PBMC人源化结肠癌模型。当肿瘤体积达到～95mm 3时，挑选个体肿瘤体积适中的小鼠入组，将动物按肿瘤体积使用Study DirectorTM(版本号3.1.399.19，供应商Studylog System,Inc.,S.San Francisco,CA,USA)进行随机分组，分配到5个实验组(G1为PBS对照组、G2为对照抗体anti-Hel hIgG1组、G3为抗TNFR2抗体001-Hu3-hIgG1组、G4为抗TNFR2抗体219-Hu1-hIgG1组、G5为Keytruda组)，两个供体donor每组5-7只，用于体内药效学研究。分组(第13天)当天开始给药。给药剂量均为10mg/kg(与图中的计量单位mpk同一含义，下同)，每周腹腔注射给药两次。每周固定时间测量肿瘤体积和体重两次。肿瘤体积(tumor volume,TV)的计算公式为：V＝1/2×a×b 2。其中a和b分别表示长和宽。在donor#193中，肿瘤接种后第29天，与对照抗体组相比，219-Hu1组的肿瘤增长被显著抑制，且具有统计学差异(P<0.0001)，其他组与对照抗体组相比无统计学差异。说明在donor#193中，抗TNFR2抗体219-Hu1具有显著的药效(图16中A)。在donor#272中，肿瘤接种后第29天，与对照抗体组相比，001-Hu3、219-Hu1、Keytruda组的肿瘤增长均被显著抑制，且具有统计学差异(P<0.0001)。说明在donor#272中，Keytruda和抗TNFR2抗体001-Hu3、219-Hu1均有药效(图16中B)。
实验过程中对照组和给药组小鼠均出现体重下降，且都有小鼠死亡，经兽医尸检结果推测小鼠死亡为注射PBMC后引起的宿主免疫排斥(GvHD)反应。测试药001-Hu3、219-Hu1和阳性药Keytruda在10mg/kg测试剂量下，动物没有表现明显的药物毒性，治疗可以耐受(图16中C-D)。
在最后一次给药后48h之后，分别选取G2对照抗体组、G3抗TNFR2抗体001-Hu3组、G4抗TNFR2抗体219-Hu1组的小鼠进行肿瘤浸润免疫细胞(TIL)分离(Miltenyi，货号 130-095-929)，进行FACS的免疫分型，并加入123 count eBeads(eBioscience，货号01-1234-42)进行细胞绝对计数，所有细胞群体的画门依据都为FMO对照(图17中A)。实验结果显示，与对照抗体组相比，donor#193抗TNFR2抗体治疗组CD4+T细胞比例降低，但数目无显著性差异,donor#272抗TNFR2抗体治疗组CD4+T细胞比例与数目均无显著性差异(图17中B-C,E-F)；与对照抗体组相比，donor#193抗TNFR2抗体治疗组CD8+T细胞数目比例增加，但数目无显著性差异，CD8+T细胞比例无显著性差异，但经001-Hu3抗体治疗组CD8+T细胞数目显著增加(图17中B-C,E-F)；两个donor的结果均显示抗TNFR2抗体治疗组的Treg(CD4+CD25+Foxp3+细胞)细胞比例和数目均显著低于对照抗体组，且具有显著性差异(图17中B-C,E-F)；同时抗TNFR2抗体治疗组的总CD8+T细胞数与Treg细胞数的比值(CD8+T/Treg)也明显高于对照抗体组，且具有统计学差异(图17中D，G)。以上的数据显示抗TNFR2抗体治疗后可显著提高TIL中CD8+T/Treg比例从而抑制肿瘤生长。
实施例17 219-Hu1-hIgG1单克隆抗体与抗鼠PD-L1单克隆抗体在TNFR2人源化BALBc小鼠模型中的联用药效检测
利用在表达人TNFR2的基因改造BALB/c小鼠(TNFR2 HuGEMM)皮下接种人TNFR2基因改造的CT26.1C03结肠癌细胞(hTNFR2 KI-CT26)的动物模型评价219-Hu1-hIgG1抗体和抗鼠PD-L1(anti-mPD-L1，BioXcell，BE0101)的联用药效实验。小鼠右侧皮下接种5×10 5CT26.1C03结肠癌细胞(设为实验第0天)。当肿瘤体积达到～100mm 3时，挑选个体肿瘤体积适中的小鼠入组，将动物按肿瘤体积使用基于EXCEL随机数的随机分组方法，分配到5个实验组(G1为对照组、G2为anti-mPD-L1 10mg/kg抗体组、G3为219-Hu1-hIgG1 10mg/kg抗体组、G4为219-Hu1-hIgG1 30mg/kg抗体组、G5为219-Hu1-hIgG1 10mg/kg+anti-mPD-L1 10mg/kg抗体联用组)，每组各10只动物，用于体内药效学研究，分组(第10天)当天开始给药。每周腹腔注射给药两次。每周固定时间测量肿瘤体积和体重三次。
在肿瘤接种后第18天(即给药第8天)，与对照组(G1)相比，G2 anti-mPD-L1 10mg/kg组、G3 219-Hu1-hIgG1 10mg/kg组和G4 219-Hu1-hIgG1 30mg/kg组的TGI TV值分别为35.1％，28.2％和63.8％，这三个治疗组的肿瘤体积增长均被显著抑制(G2vs.G1，P值<0.0001；G3vs.G1，P值＝0.0002；G4vs.G1，P值<0.0001)，219-Hu1-hIgG1 10mg/kg和anti-mPD-L1 10mg/kg的TGI TV值相当，但是219-Hu1-hIgG1 30mg/kg的TGI TV值要显著高于219-Hu1-hIgG1 10mg/kg和anti-mPD-L1 10mg/kg组(G2vs.G4，P值＝0.0002；G3vs.G4，P值<0.0001)。以上结果说明219-Hu1-hIgG1在10～30mg/kg剂量范围内存在剂量依赖效应，肿瘤体积随剂量增加而减小，详情见表11、图18。
在肿瘤接种后第18天，与对照组(G1)相比，G5 219-Hu1-hIgG1 10mg/kg+anti-mPD-L1 10mg/kg联合给药组的肿瘤体积增长也显著被抑制(TGI TV＝66.5，vs.G1，P值<0.0001)；且G5 219-Hu1-hIgG1 10mg/kg+anti-mPD-L1 10mg/kg联合给药组的平均瘤体积也显著地小于G3 219-Hu1-hIgG1 10mg/kg和G2 anti-mPD-L1 10mg/kg的单药组(TGI TV G5vs.G3vs.G2＝66.5％vs.28.2％vs.35.1％；P值G5vs.G2<0.0001，P值G5vs.G3<0.0001)，说明当使用219-Hu1-hIgG1抗体与anti-mPD-L1抗体联合给药，相比两个单药组，更能抑制肿瘤的生长且有统计学差异(****，p<0.0001，单药组vs.联合给药组)。详情见表11、图18。
表11. 219-Hu1-hIgG1和/或anti-mPD-L1对CT26.1C03同种移植瘤模型的抑瘤药效评价(基 于接种后第18天肿瘤体积计算得出)
Figure PCTCN2022074228-appb-000023
注：
a.平均值±SD。
b.相对肿瘤增长率T/C％＝T RTV/C RTV×100％(T RTV：治疗组平均RTV；C RTV：阴性对照组平均RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝V t/V 0，其中V 0是分组给药时(即PO-D0)测量所得肿瘤体积，V t为某一次测量时的肿瘤体积。
c.TGI TV(％)＝[1-(Ti-T0)/(Vi-V0)]×100％计算(Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值)。
d.数据采用Two-way ANOVA分析，vs.G1Veh，p<0.05为具有显著性差异，***:p<0.001,****:p<0.0001。
e.数据采用Two-way ANOVA，vs.G5联合给药组，p<0.05为具有显著性差异，****:p<0.0001。
f.数据采用Two-way ANOVA，vs.G4 219-Hu1-hIgG1 30mpk，p<0.05为具有显著性差异，***:p<0.001，****:p<0.0001。
各组实验动物在给药期间活动和进食状态良好，体重均有一定程度的上升。各组在实验周期内的观察中，均没有发现动物精神状况、活动状况以及体表观察的变差，动物均没有表现出药物毒性，安全性较好，说明219-Hu1-hIgG1抗体以及联合给药219-Hu1-hIgG1+anti-mPD-L1抗体的安全性较高，详情见图19-20、表12。
表12. 219-Hu1-hIgG1和/或anti-mPD-L1对CT26.1C03同种移植瘤模型的小鼠体重的影响
Figure PCTCN2022074228-appb-000024
注:平均值±SD。
在肿瘤接种后的第18天，各组动物安乐死后，对肿瘤进行分离和称重，与对照组(G1)相比，G2 anti-mPD-L1 10mg/kg组的肿瘤重量显著降低(TGI TW＝37.3％，P值0.0087)；G3 219-Hu1-hIgG1 10mg/kg组的肿瘤重量要低于对照组，但是不存在统计学差异(TGI TW＝25.9％，P值0.0830)；G4 219-Hu1-hIgG1 30mg/kg组的肿瘤重量也显著低于对照组(TGI TW＝47.9％，P值0.0013)；G5 219-Hu1-hIgG1 10mg/kg+anti-mPD-L1 10mg/kg联合给药组的肿瘤重量也 显著低于对照组(TGI TW＝43.0％，P值0.0042)；219-Hu1-hIgG1在两个剂量下的瘤重有剂量依赖效应，肿瘤重量随着给药剂量的增加而降低；联合给药组肿瘤在重量上小于各单药组(G2、G3)的瘤重，但是没有统计学差异(图21，表13)。
表13. 219-Hu1-hIgG1和/或anti-mPD-L1对CT26.1C03同种移植瘤模型的实验终点的抑瘤药效评价(基于接种后第18天肿瘤重量计算得出)
Figure PCTCN2022074228-appb-000025
注：
a.平均值±SD。
b.TGI Tw(％)＝[1-Ti/Vi]×100％计算(Ti：治疗组在给药第i天的肿瘤重量均值，Vi：溶剂对照组在给药第i天的肿瘤重量均值)。
c.数据采用One-way ANOVA方法分析，vs.G1Veh，p<0.05为具有显著性差异，**:p<0.01

Claims (23)

  1. 一种特异性结合TNFR2的人源化抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)；
    (1)所述重链可变区包含：
    a.选自SEQ ID NO.19～27任一所示的VH序列，
    b.选自与SEQ ID NO.19～27任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH序列，或，
    c.选自与SEQ ID NO.19～27任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸插入、缺失和/或替换的VH序列；
    (2)所述轻链可变区包含：
    a.选自SEQ ID NO.28～37任一所示的VL序列，
    b.选自与SEQ ID NO.28～37任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VL序列，或，
    c.选自与SEQ ID NO.28～37任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸插入、缺失和/或替换的VL序列；
    可选地，所述氨基酸插入、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
    可选地，所述替换为保守氨基酸的替换。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述重链可变区包含与SEQ ID NO.19～27任一序列相比完全一致的CDR，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH序列；和/或所述轻链可变区包含与SEQ ID NO.28～37任一序列相比完全一致的CDR，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VL序列。
  3. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述重链可变区和轻链可变区选自如下组：
    (1)具有SEQ ID NO.19所示的VH和SEQ ID NO.28所示的VL；
    (2)具有SEQ ID NO.20所示的VH和SEQ ID NO.29所示的VL；
    (3)具有SEQ ID NO.19所示的VH和SEQ ID NO.30所示的VL；
    (4)具有SEQ ID NO.20所示的VH和SEQ ID NO.31所示的VL；
    (5)具有SEQ ID NO.19所示的VH和SEQ ID NO.31所示的VL；
    (6)具有SEQ ID NO.21所示的VH和SEQ ID NO.32所示的VL；
    (7)具有SEQ ID NO.22所示的VH和SEQ ID NO.32所示的VL；
    (8)具有SEQ ID NO.23所示的VH和SEQ ID NO.32所示的VL；
    (9)具有SEQ ID NO.24所示的VH和SEQ ID NO.32所示的VL；
    (10)具有SEQ ID NO.25所示的VH和SEQ ID NO.32所示的VL；
    (11)具有SEQ ID NO.26所示的VH和SEQ ID NO.33所示的VL；
    (12)具有SEQ ID NO.27所示的VH和SEQ ID NO.34所示的VL；
    (13)具有SEQ ID NO.27所示的VH和SEQ ID NO.35所示的VL；
    (14)具有SEQ ID NO.27所示的VH和SEQ ID NO.33所示的VL；
    (15)具有SEQ ID NO.27所示的VH和SEQ ID NO.36所示的VL；
    (16)具有SEQ ID NO.26所示的VH和SEQ ID NO.34所示的VL；
    (17)具有SEQ ID NO.27所示的VH和SEQ ID NO.37所示的VL；
    (18)具有与上述(1)～(17)任一序列组合相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
    (19)具有与上述(1)～(17)任一项序列组合相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸插入、缺失和/或替换的VH和VL组合；或，
    (20)具有与上述(1)～(17)任一项序列组合相比CDR完全一致，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
    可选地，所述氨基酸插入、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
    可选地，所述替换为保守氨基酸的替换。
  4. 根据权利要求1～3任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含：
    CDR1-VH选自SEQ ID NO.1、7、或13；
    CDR2-VH选自SEQ ID NO.2、8、或14；
    CDR3-VH选自SEQ ID NO.3、9、或15；
    CDR1-VL选自SEQ ID NO.4、10、或16；
    CDR2-VL选自SEQ ID NO.5、11、或17；和
    CDR3-VL选自SEQ ID NO.6、12、或18。
  5. 根据权利要求1～4任一项所述的抗体或其抗原结合片段，其特征在于，其与人TNFR2结合的解离常数(KD)不大于7nM，与食蟹猴TNFR2结合的解离常数(KD)不大于5nM。
  6. 根据权利要求1～5任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含或不包含重链恒定区和/或轻链恒定区；
    优选地，所述重链恒定区包含全长重链恒定区或其片段，所述片段选自CH1结构域、Fc结构域或CH3结构域；
    优选地，所述重链恒定区和/或轻链恒定区为人重链恒定区和/或人轻链恒定区；
    优选地，所述重链恒定区可选自IgG重链恒定区，例如IgG1重链恒定区、IgG2重链恒定区、IgG3重链恒定区或IgG4重链恒定区；
    优选地，所述重链恒定区是人IgG1重链恒定区、人IgG2重链恒定区、人IgG3重链恒定区或人IgG4重链恒定区。
  7. 根据权利要求1～6任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、Fab、Fab’、F(ab’) 2、Fd、Fv、scFv、或双抗体(diabody)。
  8. 根据权利要求1～7任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段通过或不通过接头(linker)偶联有另一分子；优选地，所述另一分子选自治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
  9. 根据权利要求1～8任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段：
    (1)特异性结合细胞表面表达TNFR2的细胞；
    (2)特异性结合Treg细胞；
    (3)抑制TNFα与TNFR2蛋白的结合；
    (4)抑制TNFα与细胞表面表达的TNFR2的结合；
    (5)抑制TNFα介导的Treg的增殖和/或Treg功能；
    (6)抑制TNFα介导的IκBα降解；
    (7)抑制Treg对Tcon细胞增殖的抑制；
    (8)介导针对TNFR2表达细胞的ADCC功能；
    (9)提升肿瘤浸润免疫细胞TIL中CD8+T/Treg细胞比例；或/和,
    (10)抑制肿瘤生长。
  10. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子至少包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含权利要求1～9任一项所述的抗体或其抗原结合片段，所述第二抗原结合模块特异性结合TNFR2以外的其他抗原或结合与第一抗原结合模块不同的TNFR2抗原表位；
    优选地，所述其他抗原选自CD3、CD4、CD5、CD8、CD14、CD15、CD16、CD16A、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD70、CD74、CD80、CD86、CD126、CD138、B7、PD-1、PD-L1、PD-2、CTLA4、PRVIG、TIGHT、HAS、CLDN18.2、MSLN、MUC、Ia、HLA-DR、腱生蛋白、EGFR、VEGF、P1GF、ED-B纤连蛋白、癌基因产物、IL-2、IL-6、IL-15、IL-21、TRAIL-R1或TRAIL-R2；
    优选地，所述多特异性抗体为双特异性抗体、三特异性抗体或四特异性抗体。
  11. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域；所述细胞外抗原结合结构域包含权利要求1～9任一项所述TNFR2人源化抗体或其抗原结合片段，或包含权利要求10所述多特异性抗原结合分子。
  12. 一种免疫效应细胞，其特征在于，所述免疫效应细胞包含权利要求11所述嵌合抗原受体或包含编码权利要求11所述嵌合抗原受体的核酸片段；
    优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
    优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
  13. 一种分离的核酸片段，其特征在于，所述核酸片段编码权利要求1～9任一项所述的抗体或其抗原结合片段、权利要求10所述的多特异性抗原结合分子或权利要求11所述的嵌合抗原受体。
  14. 一种载体(vector)，其特征在于，所述载体包含权利要求13所述核酸片段。
  15. 一种宿主细胞，其特征在于，所述宿主细胞包含权利要求14所述载体；优选地，所述细胞为原核细胞或真核细胞，例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)；优选地，所述细胞缺乏岩藻糖基转移酶，更优选地，所述岩藻糖基转移酶是FUT8。
  16. 一种制备权利要求1～9任一项所述抗体或其抗原结合片段或权利要求10所述多特异性抗原结合分子的方法，其特征在于，所述方法包括培养权利要求15所述的宿主细胞，以及分离所述宿主细胞表达的抗体或其抗原结合片段或多特异性抗原结合分子。
  17. 一种制备权利要求12所述免疫效应细胞的方法，其特征在于，所述方法包括将编码权利要求11所述的嵌合抗原受体(CAR)的核酸片段导入所述免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达权利要求11所述CAR。
  18. 一种药物组合物，其特征在于，所述组合物包含权利要求1～9任一项所述的抗体或其抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的核酸片段、权利要求14所述的载体或权利要求15所述的细胞、权利要求16或17所述方法制备获得的产品；
    优选地，所述组合物还包含药学上可接受的载体(carrier)、稀释剂或助剂；
    优选地，所述组合物还包含额外的抗肿瘤剂、免疫治疗剂或免疫抑制剂；
    优选地，所述额外的抗肿瘤剂选自PD-1抗体、PD-L1抗体或CTLA-4抗体。
  19. 权利要求1～9任一项所述的抗体或其抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的核酸片段、权利要求14所述的载体、权利要求15所述的细胞、权利要求16或17所述方法制备获得的产品、和/或权利要求18所述的药物组合物在制备治疗和/或预防免疫异常相关疾病的药物中的用途；
    优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；
    优选地，所述疾病是癌症或自身免疫性疾病；
    优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转 移性黑色素瘤；
    优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力、IgG4相关性疾病。
  20. 一种治疗和/或预防免疫异常相关疾病的方法，其特征在于，所述方法包括向受试者施用有效量的权利要求1～9任一项所述的抗体或其抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的核酸片段、权利要求14所述的载体、权利要求15所述的细胞、权利要求16或17所述方法制备获得的产品、和/或权利要求18所述的药物组合物；
    优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；
    优选地，所述疾病是癌症或自身免疫性疾病；
    优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；
    优选地，所述自身免疫性疾病选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力、IgG4相关性疾病。
  21. 权利要求19所述用途或权利要求20所述的方法，其还包括向治疗和/或预防的对象施用额外的抗肿瘤治疗剂，例如化疗剂、靶向治疗剂和免疫治疗剂，包括PD-1/PD-L1治疗剂，如抗PD-1/PD-L1抗体、抗CTLA-4治疗剂如抗CTLA-4抗体；
    优选地，所述额外的抗肿瘤治疗剂选自PD-1/PD-L1治疗剂；
    优选地，所述PD-1/PD-L1治疗剂选自PD-1抗体或PD-L1抗体。
  22. 权利要求1～9任一项所述的抗体或其抗原结合片段、权利要求10所述的多特异性抗原结合分子、权利要求11所述的嵌合抗原受体、权利要求12所述的免疫效应细胞、权利要求13所述的核酸片段、权利要求14所述的载体、权利要求15所述的细胞、权利要求16或17所述方法制备获得的产品、和/或权利要求18所述的药物组合物，其特征在于，用于治疗和/或预防免疫异常相关疾病；
    优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；
    优选地，所述免疫异常相关疾病是癌症或自身免疫性疾病；
    优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；
    优选地，所述自身免疫性疾病选自类风湿性关节炎、多发性硬化症、系统性硬化症、视神经脊髓炎谱系病、系统性红斑狼疮、重症肌无力、IgG4相关性疾病；
    优选地，其还包括向治疗和/或预防的对象施用额外的抗肿瘤治疗剂，例如化疗剂、靶向 治疗剂和免疫治疗剂，包括PD-1/PD-L1治疗剂，如抗PD-1/PD-L1抗体、抗CTLA-4治疗剂如抗CTLA-4抗体。
  23. 一种体外检测样品中TNFR2的方法，其包括使怀疑含有TNFR2的样品与权利要求1至9任一项所述的抗体或其抗原结合片段相接触的步骤。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023125483A1 (zh) * 2021-12-28 2023-07-06 山东先声生物制药有限公司 一种抗tnfr2抗体药物组合物
WO2024037626A1 (zh) * 2022-08-19 2024-02-22 盛禾(中国)生物制药有限公司 一种结合tnfr2和4-1bb的双特异性抗体
WO2024037628A1 (zh) * 2022-08-19 2024-02-22 盛禾(中国)生物制药有限公司 一种双特异性抗体及其应用
WO2024037627A1 (zh) * 2022-08-19 2024-02-22 盛禾(中国)生物制药有限公司 一种双特异性抗体及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109476745A (zh) * 2016-05-13 2019-03-15 综合医院公司 拮抗性抗肿瘤坏死因子受体超家族抗体
WO2020089474A1 (en) * 2018-11-01 2020-05-07 Bioinvent International Ab Novel antagonistic anti tnfr2 antibody molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109476745A (zh) * 2016-05-13 2019-03-15 综合医院公司 拮抗性抗肿瘤坏死因子受体超家族抗体
WO2020089474A1 (en) * 2018-11-01 2020-05-07 Bioinvent International Ab Novel antagonistic anti tnfr2 antibody molecules

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
GILLIES ET AL., J IMMUNOL METHODS, vol. 125, 1985, pages 191 - 202
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE
MORRISON, SCIENCE, vol. 229, no. 4719, 1985, pages 1202 - 7
OI ET AL., BIO TECHNIQUES, vol. 4, 1986, pages 214 - 221
WAHL ET AL., J.NUCL.MED, vol. 24, 1983, pages 316
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
ZARAGOZA B ET AL., NAT MED, vol. 22, no. 1, January 2016 (2016-01-01), pages 16 - 7

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023125483A1 (zh) * 2021-12-28 2023-07-06 山东先声生物制药有限公司 一种抗tnfr2抗体药物组合物
WO2024037626A1 (zh) * 2022-08-19 2024-02-22 盛禾(中国)生物制药有限公司 一种结合tnfr2和4-1bb的双特异性抗体
WO2024037628A1 (zh) * 2022-08-19 2024-02-22 盛禾(中国)生物制药有限公司 一种双特异性抗体及其应用
WO2024037627A1 (zh) * 2022-08-19 2024-02-22 盛禾(中国)生物制药有限公司 一种双特异性抗体及其应用

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