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WO2022166846A1 - 抗tnfr2抗体及其用途 - Google Patents

抗tnfr2抗体及其用途 Download PDF

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Publication number
WO2022166846A1
WO2022166846A1 PCT/CN2022/074755 CN2022074755W WO2022166846A1 WO 2022166846 A1 WO2022166846 A1 WO 2022166846A1 CN 2022074755 W CN2022074755 W CN 2022074755W WO 2022166846 A1 WO2022166846 A1 WO 2022166846A1
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Prior art keywords
antibody
amino acid
seq
acid sequence
tnfr2
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PCT/CN2022/074755
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English (en)
French (fr)
Inventor
黎一鸣
陈炳良
荆华
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信达生物制药(苏州)有限公司
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Priority to EP22749125.5A priority Critical patent/EP4289865A1/en
Priority to US18/263,455 priority patent/US20240026019A1/en
Priority to CA3209827A priority patent/CA3209827A1/en
Priority to AU2022217845A priority patent/AU2022217845A1/en
Priority to CN202280013275.1A priority patent/CN116848142A/zh
Priority to JP2023547245A priority patent/JP2024505987A/ja
Publication of WO2022166846A1 publication Critical patent/WO2022166846A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the antibody TNFR2 antibody, its preparation method, composition and use.
  • the present invention also provides methods for treating TNFR2-related diseases and/or disorders, such as cancer.
  • Type II tumor necrosis factor receptor (TNFR2, also known as CD120b or p75 or TNFRSF1B), is a member of the tumor necrosis factor (TNF) receptor superfamily.
  • TNF tumor necrosis factor
  • TNFR2 is highly expressed on the surface of various types of cancer cells and regulatory T cells (Treg) that can infiltrate into tumors and suppress the activity of the immune system.
  • Treg regulatory T cells
  • TNF ⁇ By binding to TNFR2, TNF ⁇ can activate downstream NFkB signaling, resulting in enhanced Treg cell proliferation.
  • a high abundance of TNFR2-positive Treg cells has been found in the tumor microenvironment in various human and murine cancers. Therefore, treating cancer by targeting Treg cells in the tumor microenvironment and TNRF2 on tumor cells is a promising therapeutic option.
  • WO2017/083525 discloses some antagonistic TNFR2 antibodies and proposes their use in cancer therapy.
  • TNFR2 In view of the potential target role of TNFR2 in tumor immune response, there is a need in the art to develop new TNFR2 antibodies, especially anti-TNFR2 antibodies with strong blocking effect and direct tumor killing activity, for disease treatment, especially is cancer treatment.
  • the present inventors provided new anti-TNFR2 antibodies through intensive research.
  • the anti-TNFR2 antibody of the present invention has strong TNFR2 blocking activity, and some molecules have a new blocking mechanism, which can directly inhibit the multimerization of TNFR2 without blocking the combination of TNF ⁇ and receptor TNFR2, thereby inhibiting TNF ⁇ /TNFR2 activation of signaling pathways.
  • the anti-TNFR2 antibody of the present invention shows good anti-tumor efficacy results in animal experiments.
  • the present invention provides an anti-TNFR2 antibody or antigen-binding fragment thereof comprising: one of the heavy chain variable regions set forth in SEQ ID NOs: 19, 21, 23, 25, 27, 29, 64 HCDR1, 2 and 3 sequences, and/or LCDR1, 2 and 3 sequences of the light chain variable region set forth in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 31, 65, or the CDR sequences Combination variant.
  • the anti-TNFR2 antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:
  • (vi) has anti-tumor activity, such as inhibiting tumor growth.
  • the invention also provides anti-TNFR2 antibodies, or antigen-binding fragments thereof, that bind the same or overlapping exemplified antibodies of the invention (eg, antibodies having combinations of antibody VH and VL sequences listed in Table B below) Epitope and/or competitive binding to TNFR2, and/or inhibition (eg, competitive inhibition) of exemplary antibodies of the invention.
  • anti-TNFR2 antibodies or antigen-binding fragments thereof, that bind the same or overlapping exemplified antibodies of the invention (eg, antibodies having combinations of antibody VH and VL sequences listed in Table B below) Epitope and/or competitive binding to TNFR2, and/or inhibition (eg, competitive inhibition) of exemplary antibodies of the invention.
  • the invention provides nucleic acids encoding antibodies of the invention or antigen-binding fragments thereof, vectors comprising the nucleic acids, and host cells comprising the vectors.
  • the present invention provides methods of making the antibodies or antigen-binding fragments thereof of the present invention.
  • the present invention provides immunoconjugates, pharmaceutical compositions and combination products comprising the antibodies of the present invention.
  • the present invention provides in vivo and in vitro methods and uses using the TNFR2 antibodies or antigen-binding fragments thereof of the present invention, including, for example, but not limited to, for blocking and/or antagonizing TNFR-2 and its ligands in vivo and in vitro TNF ⁇ binds, and/or blocks and/or antagonizes biological activities resulting from the binding, such as activation of the TNFR2 receptor and/or proliferation of Treg cells; and/or for killing surface-expressing TNFR2 in vivo and in vitro tumor cells.
  • the present invention also provides methods and uses for the prevention or treatment of TNFR2-related diseases using the antibodies of the present invention or antigen-binding fragments thereof, including, but not limited to, tumors, such as colon cancer or chronic myeloid leukemia.
  • the antibodies of the invention may be used as the sole active agent, or may be administered in combination with other therapies or therapeutic agents.
  • the present invention also provides a method and kit for detecting TNFR2 in a sample using the antibody or antigen-binding fragment thereof of the present invention.
  • Figure 1 shows flow cytometry to determine the binding ability of hybridoma antibodies to human TNFR2-overexpressing stably transfected cell line hTNFR2 CHO-S.
  • Figure 2 shows flow cytometry to determine the binding ability of hybridoma antibodies to cynomolgus monkey TNFR2 stably transfected cell line cTNFR2 CHO-S.
  • Figure 3 shows the blocking effect of hybridoma antibodies on TNF ⁇ /TNFR2 detected by ELISA.
  • Figure 4 shows flow cytometry to determine the binding ability of the chimeric antibody to the human TNFR2 overexpressing stably transfected cell line hTNFR2 CHO-S.
  • Figure 5 shows flow cytometry to determine the binding ability of the chimeric antibody to cTNFR2 CHO-S overexpressing cynomolgus monkey TNFR2 stably transfected cell line.
  • Figure 6 shows the blocking effect of chimeric antibodies on TNF ⁇ /TNFR2 detected by ELISA.
  • Figure 7 shows the use of Jurkat TNFR2 NF- ⁇ B luciferase reporter gene assay to detect the inhibitory effect of chimeric antibodies on TNF ⁇ -activated TNFR2 signaling pathway.
  • Figure 8 shows the binding ability of humanized antibody to human hTNFR2 CHO-S stably transfected cell line determined by flow cytometry.
  • Figure 9 shows the blocking effect of humanized antibody on TNF ⁇ /TNFR2 detected by ELISA.
  • Figure 10 shows the use of the Jurkat TNFR2 NF- ⁇ B luciferase reporter gene assay to detect the inhibitory effect of humanized antibodies on TNF ⁇ -activated TNFR2 signaling pathway.
  • Figure 11 shows the binding ability of humanized antibodies to human K562 CML cell line (A) and primary Treg cells (B) determined by flow cytometry.
  • Figure 12 shows the use of the Jurkat ADCC luciferase reporter gene assay to detect the activation of the ADCC signaling pathway by humanized antibodies.
  • Figure 13 shows the K562 matrigel NOD/SCID animal model to detect the anti-tumor effect of TNFR2 antibody.
  • Figure 14 shows the anti-tumor effect of TNFR2 antibody detected by subcutaneous inoculation of C57 animal model with MC38-TNFR2.
  • Figure 15 shows the CDR sequences of exemplary chimeric and humanized antibodies of the invention.
  • Figure 16 shows the VH and VL sequences of exemplary chimeric and humanized antibodies of the invention.
  • the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
  • the term “comprising” or “comprising” is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps.
  • reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
  • antibody refers to a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an antigen.
  • the term encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, single- or multi-chain antibodies, monospecific or multispecific antibodies (eg, bispecific antibodies), fully human antibodies, or Chimeric or humanized antibodies, full-length antibodies and antibody fragments, so long as they exhibit the desired antigen-binding activity.
  • a “whole antibody” (used interchangeably herein with “full length antibody”, “complete antibody” and “intact antibody”) comprises at least two heavy (H) chains and two light chains ( L).
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the variable region is the domain of the heavy or light chain of an antibody that is involved in binding the antibody to its antigen.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.
  • the light chains of antibodies can be assigned to one of two types, termed kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of their constant domains.
  • the heavy chains of antibodies can be divided into mainly 5 different classes depending on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses such as , IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2.
  • the heavy chain constant regions corresponding to the different antibody types are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the term "isotype” refers to the type of antibody determined by the constant region of the antibody heavy chain. See, eg, Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd ed., Raven Press, N.Y. (1989)) (which is hereby incorporated by reference in its entirety for all purposes).
  • antigen-binding portion of an antibody refers to a molecule that is not an intact antibody, comprising the part of the antigen.
  • the antigen-binding portion of an antibody typically comprises amino acid residues from "complementarity determining regions" or "CDRs.”
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab') 2 , Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody ( tetrabody), minibody, single domain antibody (sdAb).
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, eg, an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody .
  • humanized antibody refers to an antibody in which CDR sequences derived from the germline of other mammalian species, such as mouse, are attached to human framework sequences. Additional framework region modifications can be made within the human framework sequences, and/or additional amino acid modifications within the CDR sequences, eg, for affinity maturation of antibodies.
  • an “isolated” antibody is one that has been separated from components of its natural environment.
  • the antibody is purified to greater than 95% or 99% purity by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reaction phase HPLC) to determine.
  • electrophoresis eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography eg, ion exchange or reaction phase HPLC
  • epitope refers to the region of an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein.
  • TNRF2 refers to "Tumor Necrosis Factor Receptor 2", also known as TNFRSF1B and CD120b.
  • This receptor a member of the tumor necrosis factor receptor superfamily (TNFRSF), acts as a cell surface membrane-bound receptor that can be expressed on cancer cells and tumor-infiltrating Treg cells.
  • TNFRSF tumor necrosis factor receptor superfamily
  • TNFR2 can regulate the transcription of genes that promote cell survival and proliferation.
  • the expression encompasses TNFR2 and its variants, isoforms, homologues, and species homologues.
  • the TNFR2 is a TNFR2 derived from a human, for example, the human TNFR2 shown in SEQ ID NO:60, or at least 95%, even at least 96%, 97% with SEQ ID NO:60 , 98%, or 99% amino acid sequence identity to human TNFR2.
  • the TNFR2 is a monkey-derived TNFR2, eg, human TNFR2 as set forth in SEQ ID NO:61, or at least 95%, even at least 96% with SEQ ID NO:61,97 %, 98%, or 99% amino acid sequence identity to human TNFR2.
  • TNFR2 may also include fragments of TNFR2, such as fragments comprising an extracellular domain, eg, fragments that retain the ability to bind to any of the antibodies of the invention.
  • TNF ⁇ refers to the natural agonist ligand of TNFR2, "Tumor Necrosis Factor ⁇ ".
  • TNFRl refers to "tumor necrosis factor receptor 1", eg human TNFRl under UniProtKB accession number P19438.
  • the term “specifically binds” means that an antibody binds selectively or preferentially to an antigen. If in bioluminescence interferometry, the antibody is at about 5x 10-7 M or less, about 1x 10-7 M or less, about 5x 10-8 M or less, about 1x 10-8 M or less, An antibody that "specifically binds to human TNFR2 " with a KD of about 5x 10-9 M or less, which binds to human TNFR2.
  • antibodies that specifically bind human TNFR2 may be cross-reactive with TNFR2 proteins from other species.
  • antibodies specific for human TNFR2 in some embodiments, can cross-react with TNFR2 proteins of non-human species. In other embodiments, antibodies specific for human TNFR2 may be fully specific for human TNFR2 without species or other types of cross-reactivity, or only with certain species of TNFR2.
  • cross-reactivity refers to the ability of an antibody to bind TNFR2 from different species.
  • the antibodies described herein that bind human TNFR2 may also bind TNFR2 from other species (eg, cynomolgus TNFR2).
  • Methods for determining cross-reactivity include those described in the Examples as well as standard assays known in the art, eg, by using bio-light interference, or flow cytometry techniques.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant ( kdis and kon , respectively ) .
  • KD equilibrium dissociation constant
  • Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • high affinity with respect to IgG antibodies means that the antibody has 1 x 10-7 M or less, preferably 5 x 10-8 M or less, more preferably about 1 x 10-8 M or less, even more preferably A KD of approximately 5x 10-9 M or lower, bound to the target antigen.
  • “high affinity” binding can vary with antibody isotype.
  • “high affinity” refers to an antibody having a KD of 1 x 10-6 M or less, preferably 1 x 10-7 M or less, more preferably about 1 x 10-8 M or less.
  • an "antibody that competes for binding" with a reference antibody that binds an antigen such as TNFR2 refers to an antibody that blocks 50% or more of the binding of the reference antibody to an antigen (eg, TNFR2) in a competition assay, and in turn, A reference antibody blocks 50% or more of the binding of the antibody to an antigen (eg, TNFR2) in a competition assay.
  • Exemplary competition tests are described in: "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • Antibodies competing for binding may bind the same epitope region, eg, the same epitope, adjacent epitopes, or overlapping epitopes, as the reference antibody.
  • An antibody that inhibits (eg competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of said reference antibody to its antigen . Conversely, the reference antibody inhibits more than 50%, 60%, 70%, 80%, 90% or 95% of the binding of the antibody to its antigen. Binding of an antibody to its antigen can be measured by affinity (eg, equilibrium dissociation constant). Methods for determining affinity are known in the art.
  • An antibody that exhibits the same or similar binding affinity and/or specificity as a reference antibody refers to an antibody capable of binding at least 50%, 60%, 70%, 80%, 90% or 95% or more of the reference antibody Affinity and/or specificity. This can be determined by any method known in the art for determining binding affinity and/or specificity.
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc-regions and variant Fc-regions.
  • the human IgG heavy chain Fc-region extends from Cys226 of the heavy chain or from Pro230 to the carboxy terminus.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • the numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service , described in National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • effector function refers to those biological activities attributable to the Fc-region of an antibody, which vary by class of antibody.
  • Five main classes of antibodies are known to exist: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 .
  • the IgG Fc region can mediate several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and half-life/clearance rate of antibodies and antigen-antibody complexes.
  • the present invention provides variant Fc regions having amino acid residue changes in the Fc region that alter antibody effector function and methods of screening the same. For example, at least one amino acid residue can be substituted in the Fc region of an antibody, thereby altering the effector function of the antibody.
  • ADCC refers to antibody-dependent cell-mediated cytotoxicity.
  • ADCC is mainly mediated by natural killer cells (NK cells) in humans.
  • NK cells natural killer cells
  • the antibody binds to the antigen displayed on the surface of target cells, and Fc ⁇ RIIIA on the surface of NK cells recognizes the Fc region of the antibody, so that NK cells are activated to release perforin and granulolytic enzymes, resulting in lysis and apoptosis of target cells.
  • NK cells natural killer cells
  • Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in US 5,500,362 (see also, eg, Hellstrom, I. et al., Proc. Nat'l Acad. Sci.
  • nonradioactive assays eg, the ACTITM Nonradioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and Non-radioactive cytotoxicity assay (Promega, Madison, WI)
  • Useful effector cells for these assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells.
  • ADCC activity of target molecules can be assessed in vivo, eg, in animal models as disclosed in Clynes, R. et al., Proc. Nat'lAcad. Sci. USA 95 (1998) 652-656 .
  • CDC refers to complement-dependent cytotoxicity.
  • the Fc region of an antibody binds to the complement molecule C1q, which in turn forms a membrane attack complex, leading to clearance of target cells.
  • ADCP refers to antibody-dependent cell-mediated phagocytosis.
  • Fc receptor-mediated process antibody-bound target cells are phagocytosed by phagocytic cells such as macrophages, monocytes, neutrophils, and dendritic cells.
  • phagocytic cells such as macrophages, monocytes, neutrophils, and dendritic cells.
  • Fc receptors can be involved in this process. Richards et al, Mol. Cancer Ther. 7(8):2517-2527 (2008) describe in vitro assays for ADCP.
  • variant in relation to an antibody is used herein to mean, as compared to a reference antibody, comprising at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or An antibody that has an amino acid altered region of the antibody of interest with 5 amino acid substitutions, deletions, and/or insertions, wherein the variant substantially retains at least one biological property (eg, antigen binding ability) of the antibody molecule prior to the alteration.
  • the antibody region of interest can be the full length of the antibody, or a heavy chain variable region or a light chain variable region or a combination thereof, or a heavy chain CDR region(s) or a light chain CDR region(s) or a combination thereof .
  • An antibody region having amino acid changes relative to a reference antibody region is also referred to herein as a "variant" of the antibody region.
  • sequence identity refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. "Percent sequence identity” can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • the same amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • Optimal alignment to determine percent sequence identity can be achieved in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest.
  • the percent amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the reference antibody sequence, and in a preferred embodiment, performing optimal alignment according to the Kabat numbering system.
  • a comparison window ie, regions of the antibody of interest to be compared
  • the sequence identity may be distributed over the entire heavy chain variable region and/or the entire light chain variable region, or the percent sequence identity may be limited to the framework regions only, while the corresponding CDR regions The sequences remain 100% identical.
  • candidate antibodies can be identified that have amino acid changes in the region of the antibody of interest relative to the reference antibody.
  • “conservative substitution” refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid.
  • Amino acid modifications, such as substitutions can be introduced into the antibodies of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservatively substituted residues are from the conservative substitutions Table A below, preferably the preferred conservatively substituted residues shown in Table A.
  • the present invention provides antibodies or antigen-binding fragments thereof, especially humanized antibodies or antigen-binding fragments thereof, that specifically bind to TNFR2, preferably a human TNFR2 protein (eg, human TNFR2 sequence of SEQ ID NO:60, NM_001066.2).
  • the antigen-binding fragment of an antibody of the invention is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibodies such as scFv, (Fab') 2 fragments, single domain antibodies , diabody (dAb) or linear antibody.
  • an anti-TNFR2 antibody or antigen-binding fragment thereof of the invention binds to human TNFR2 with high affinity, eg, with a dissociation equilibrium constant (K D ) of less than or equal to about 50 x 10 -9 M, more preferably about 1 -30 x 10 -9 M, more preferably less than or equal to about 5 x 10 -9 M, eg about 2 nM, 1.5 nM, 1.0 nM, 0.5 nM, 0.3 nM, 0.2 nM.
  • the K D is determined by using biooptical interferometry (eg Fortebio affinity measurement).
  • the anti-TNFR2 antibody or antigen-binding fragment thereof of the invention binds to human TNFR2 with a dissociation constant, eg, as determined by Fortebio affinity measurement, of about 1-20 x 10-4 s -1 , eg, about 2x 10 -4 s -1 , about 5x 10 -4 s -1 , about 10x 10 -4 s -1 , about 15x 10 -4 s -1 , about 20x 10 -4 s -1 .
  • a dissociation constant eg, as determined by Fortebio affinity measurement
  • the anti-TNFR2 antibodies or antigen-binding fragments thereof of the invention cross-react with monkey TNFR2.
  • the antibody binds cynomolgus monkey TNFR2 with high affinity, wherein the K value (eg, by Fortebio affinity measurement) is less than or equal to about 50 x 10" 9 M, more preferably about 1-30 x 10 " 9 M, more preferably less than or equal to about 5 ⁇ 10 ⁇ 9 M.
  • the antibodies or antigen-binding fragments thereof of the invention bind to cell surface expressed TNFR2 with high affinity.
  • the cells expressing human TNFR2 on their surface are tumor cells (eg, lymphoma cells, eg, K562 cells), Treg cells (eg, human peripheral blood native CD4 + CD25 + CD127 LOW regulatory T cells), or Mammalian cells recombinantly expressing TNFR2.
  • tumor cells eg, lymphoma cells, eg, K562 cells
  • Treg cells eg, human peripheral blood native CD4 + CD25 + CD127 LOW regulatory T cells
  • Mammalian cells recombinantly expressing TNFR2 eg, the EC50 value of antibody binding to cells expressing human TNFR2 is determined by flow cytometry (eg, FACS).
  • the antibodies of the invention have cell surface TNFR2 binding EC50 values of less than 10 nM, eg, 0.1-5 nM, eg, as measured by flow cytometry on CHO cells recombinantly expressing TNFR2. In other embodiments, the antibodies of the invention have cell surface TNFR2 binding EC50 values of less than 100 pM, eg, 20-60 pM, eg, as measured by flow cytometry on K562 cells or human peripheral blood Treg cells. It will be apparent to those of skill in the art that, in some cases, the density of TNFR2 on the cell surface will affect the EC50 measurement of antibody binding to cells.
  • the antibodies or antigen-binding fragments thereof of the invention block the binding of TNFR2 to its ligand TNF[alpha].
  • the ability of an antibody to block binding of human TNFR2 (TNFR2 expressed on cells) to human TNF ⁇ can be determined by flow cytometry (eg, FACS).
  • the antibodies of the invention are capable of completely blocking the binding of human TNFR2 (TNFR2 expressed on cells) to human TNF[alpha].
  • the antibody of the invention has an IC50 value of less than about 10 nM, eg, about 1, 2, 3, 4, 5, 6 nM, as measured by FACS.
  • the antibodies of the invention or antigen-binding fragments thereof reduce the activation of the NF- ⁇ B signaling pathway caused by TNF ⁇ binding to TNFR2.
  • the ability of an antibody to reduce NF- ⁇ B signaling pathway activation by TNF ⁇ binding to TNFR2 is detected by a fluorescent reporter gene assay (eg, the Jurkat NF- ⁇ B Fluorescent Reporter Assay of the Examples) ).
  • the antibody blocks the activation of the NF- ⁇ B signaling pathway caused by the binding of TNF ⁇ to TNFR2, resulting in greater than 3-fold inhibition compared to no antibody addition.
  • the antibody has an IC50 value of less than about 0.1-10 nM.
  • binding of an antibody or antigen-binding fragment thereof of the invention to cellular TNFR2 activates the ADCC signaling pathway in the presence of NK and/or effector T cells.
  • the antibody of the present invention is capable of killing TNFR2 positive tumor cells through ADCC activity.
  • the ability of an antibody to bind to TNFR2 to elicit a fluorescent signal of ADCC is detected by a fluorescent reporter gene assay (such as the Jurkat ADCC luciferase reporter gene detection assay of the Examples).
  • the antibodies or antigen-binding fragments thereof of the invention inhibit the growth of tumors expressing human TNFR2.
  • the tumor cells are lymphoma cells expressing human TNFR2, eg, chronic myeloid leukemia cells, or solid tumor cells expressing human TNFR2, eg, colon cancer cells.
  • the antibody or antigen-binding fragment thereof of the invention elicits ADCC-dependent tumor cell killing activity.
  • the antibodies or antigen-binding fragments thereof of the invention inhibit (eg, in the tumor microenvironment) the function of TNFR2-expressing immunosuppressive regulatory Treg cells.
  • the antibodies or antigen-binding fragments thereof of the invention have at least one or preferably both of the following effects:
  • the antibodies of the invention or antigen-binding fragments thereof achieve at least 10% tumor inhibition, eg, at least 20%-50%, in recipients (eg, tumor-bearing animal models, such as tumor-bearing mice of the Examples) or higher tumor suppression rates.
  • the antibody or antigen-binding fragment thereof of the invention exhibits at least one, more preferably at least two, more preferably at least three, four, or five, even more preferably all of the above properties.
  • CDR region or “CDR” (used interchangeably herein with a hypervariable region “HVR”) is the region of amino acids in the variable region of an antibody that is primarily responsible for binding to an antigenic epitope.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • the CDRs located within the variable domains of antibody heavy chains are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the variable domains of antibody light chains are referred to as LCDR1, LCDR2 and LCDR3.
  • VH and VL sequence combinations for some exemplary antibodies of the invention are given in Table B below:
  • CDRs Kabat complementarity determining regions
  • Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • AbM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. "Contact” HVR is based on the analysis of available complex crystal structures. The residues of each of these HVRs/CDRs are described below according to different CDR determination schemes.
  • the HVR may also be an HVR sequence located at the following Kabat residue positions according to the Kabat numbering system:
  • Positions 24-36 or 24-34 in VL (LCDR1), Positions 46-56 or 50-56 (LCDR2), and Positions 89-97 or 89-96 (LCDR3); and Positions 26-35 in VH or 27-35B (HCDR1), positions 50-65 or 49-65 (HCDR2), and positions 93-102, 94-102 or 95-102 (HCDR3).
  • HVRs can also be determined based on having the same Kabat numbering positions as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • CDR or “CDR sequence” or “HVR” or “HVR sequence” encompasses HVR or CDR sequences determined in any of the ways described above.
  • a residue position in an antibody variable region refers to the numbering system according to the Kabat ( Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDR sequences of the present invention are shown in FIG. 15 .
  • Antibodies with different specificities have different CDRs.
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within CDRs are directly involved in antigen binding.
  • the minimal binding unit can be a sub-portion of a CDR.
  • the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody, as will be apparent to those skilled in the art. Accordingly, the present invention also contemplates variants of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be replaced by conservative amino acid residues.
  • the antibody of the invention has at least one, two, three, four, five or six CDRs that are identical to the corresponding CDRs in the variable region sequence of any one of the antibodies listed in Table B, or its variants.
  • the antibodies of the invention have at least one, two, or three HCDRs that are identical to, or variants of, the corresponding heavy chain CDRs in the variable region sequence of any of the antibodies listed in Table B.
  • the antibodies of the invention have at least one, two, or three LCDRs that are identical to, or variants of, the corresponding light chain CDRs in the variable region sequence of any of the antibodies listed in Table B.
  • corresponding CDRs refer to CDRs that, after optimal alignment, are located at the most similar positions to the CDRs of the reference antibody in the variable region amino acid sequence of the candidate antibody.
  • a CDR variant is a CDR that has been modified by at least one, eg 1 or 2 or 3 amino acid substitution, deletion and/or insertion, wherein the antigen binding molecule comprising the CDR variant remains substantially comprising the unmodified CDR
  • the biological property of an antigen-binding molecule eg, maintaining at least 60%, 70%, 80%, 90%, or 100% of its biological activity (eg, antigen-binding capacity).
  • each CDR may be modified individually or in combination.
  • the amino acid modifications are amino acid substitutions, especially conservative amino acid substitutions, such as the preferred conservative amino acid substitutions listed in Table A.
  • the CDR3 regions independently of the CDR1 and/or CDR2 regions, alone can determine the binding specificity of an antibody to a cognate antigen. Also, a variety of other antibodies with the same binding specificity can be generated based on common CDR3 sequences.
  • the antibodies of the invention comprise CDR3 sequences from the heavy and/or light chain variable region sequences of any of the antibodies shown in Table B, wherein the antibody is capable of specifically binding human TNFR2.
  • the antibody may also comprise CDR2s from the heavy and/or light chain variable regions of the same antibody, or CDR2s from the heavy and/or light chain variable regions of different TNFR2 antibodies.
  • the antibody may also comprise CDR1 from the heavy and/or light chain variable regions of the same antibody, or CDR1 from the heavy and/or light chain variable regions of a different TNFR2 antibody.
  • the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., , CDRs from different antibodies that bind the same TNFR2 antigen can be mixed and matched, although each antibody preferably contains VH CDRs 1, 2 and 3 and VL CDRs 1, 2 and 3) to generate other molecules of the invention that bind TNFR2.
  • Such "mixed and matched" antibodies can be tested for binding to TNFR2 using binding assays known in the art (eg, ELISA, SET, Biacore) and those described in the Examples.
  • CDR1, CDR2 and/or CDR3 sequences from a particular VH sequence are preferably replaced with structurally similar CDR sequences.
  • CDR1, CDR2 and/or CDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences.
  • "Mixing and matching" of CDRs can be performed between the antibodies shown in Table B of the invention.
  • other structurally similar CDR sequences of the antibodies shown herein can also be generated by substituting one or more VH and/or VL CDR region sequences from other different antibodies for structurally similar CDR sequences of the present invention. Antibody.
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region comprising a heavy chain complementarity determining region 3 (HCDR3) that:
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, and the antibody has a heavy chain complementarity determining region 3 (HCDR3) and a light chain complementarity determining region 3 ( LCDR3):
  • HCDR3 heavy chain complementarity determining region 3
  • LCDR3 light chain complementarity determining region 3
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH), wherein the VH comprises:
  • the antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VL), wherein the VL comprises:
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises:
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • the antibody or antigen-binding fragment thereof of the invention comprises the 3 complementarity determining regions HCDR of the heavy chain variable region, and the 3 complementarity determining regions LCDR of the light chain variable region, wherein
  • HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2
  • HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3
  • LCDR1 comprises the amino acid sequence of SEQ ID NO:4
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:5
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:6; or
  • HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:7
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:8
  • HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:9
  • LCDR1 comprises The amino acid sequence of SEQ ID NO: 10 or consists of
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 11
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 12; or
  • HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14
  • HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15
  • LCDR1 comprises The amino acid sequence of SEQ ID NO: 16 or consists of
  • LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17
  • LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof of the invention comprises 6 CDR sequences in one of the combinations listed in Table C.
  • a “variable region” or “variable domain” is the domain of an antibody's heavy or light chain that is involved in the binding of the antibody to its antigen.
  • the heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into hypervariable regions (HVRs, also known as complementarity determining regions (CDRs)) with more conserved regions (i.e., frameworks) interposed therebetween. area (FR)).
  • HVRs hypervariable regions
  • CDRs complementarity determining regions
  • FR area
  • VH or VL domain is sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen can be isolated using the VH or VL domains from antibodies that bind the antigen to screen libraries of complementary VL or VH domains (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993). ) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).
  • one or more residues in one or both of the two variable regions can be modified, eg, in one or more CDR regions and/or in one Residue modifications, particularly conservative residue substitutions, are carried out in the framework region(s), while the modified antibody substantially retains at least one biological property (eg, antigen-binding ability) of the antibody molecule before the modification.
  • residues in the CDR regions can be mutated to improve one or more binding properties (eg, affinity) of the antibody.
  • the antigen binding properties or other functional properties of the mutated antibodies can be assessed in in vitro or in vivo assays.
  • conservative substitutions are introduced.
  • framework region residues can be mutated, eg, to improve the properties of the antibody.
  • one or more framework residues can be "backmutated" to the corresponding germline sequence residues.
  • CDR grafting is another way of modifying antibody variable regions known in the art. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants can be constructed that mimic the properties of known antibodies. In this antibody variant, CDR sequences from a known antibody are grafted onto the framework regions of a different antibody with different properties.
  • the present invention relates to an anti-TNFR2 antibody, or antigen-binding fragment thereof, comprising the CDR sequences from the heavy and light chain variable regions of one of the antibodies of Table B, but with different frameworks region sequence. Framework region sequences for replacement can be obtained from public DNA databases, including germline antibody gene sequences, or from TNFR2 antibody sequences reported in the open literature.
  • germline DNA encoding human heavy and light chain variable region genes can be obtained from the GenBank database.
  • Antibody protein sequences can be compared to protein sequences in databases using sequence similarity search tools, such as Gapped BLAST.
  • sequence similarity search tools such as Gapped BLAST.
  • the framework sequence used for the substitution has structural similarity to the framework sequence of the antibody of the invention selected for alteration, e.g., has sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97% %, 98%, more than 99% of the framework sequences.
  • the humanization of antibodies can be performed in the manner of Example 9.
  • the VH and VL from an exemplary antibody of the invention can be "mixed and matched" sequences to generate other antibodies of the invention that bind TNFR2.
  • the VH sequence from a particular VH/VL pair is replaced with a structurally similar VH sequence.
  • VL sequences from a particular VH/VL pair are preferably replaced with structurally similar VL sequences.
  • Such "mixed and matched" antibodies can be tested for binding to TNFR2 using binding assays known in the art (eg, ELISA, and other assays described in the Examples section).
  • the antibody of the invention comprises, or consists of, the heavy chain variable region VH sequence of any of the antibodies listed in Table B. In yet another embodiment, the antibody of the invention comprises a variant of said VH sequence.
  • the antibody of the invention comprises, or consists of, the light chain variable region VL sequence of any one of the antibodies listed in Table B. In yet another embodiment, the antibody of the invention comprises a variant of the VL sequence.
  • the antibody of the invention comprises:
  • VH sequence comprising the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, and/or a VL sequence comprising the amino acid sequence set forth in SEQ ID NO: 20 or a variant thereof, or
  • VH sequence comprising the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof, and/or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 22 or a variant thereof, or
  • VH sequence comprising the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof, and/or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 24 or a variant thereof, or
  • VH sequence comprising the amino acid sequence set forth in SEQ ID NO: 25 or a variant thereof, and/or a VL sequence comprising the amino acid sequence set forth in SEQ ID NO: 26 or a variant thereof, or
  • VH sequence comprising the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof, and/or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 30 or 21 or a variant thereof;
  • VH sequence comprising the amino acid sequence shown in SEQ ID NO:64 or a variant thereof, and/or a VL sequence comprising the amino acid sequence shown in SEQ ID NO:65 or a variant thereof.
  • the variant of the VH sequence has at least 80%, 85%, 85%, 80%, 85%, 85%, 85%, 85%, 85%, 85%, 85% value in amino acid sequence compared to the reference VH sequence (preferably over the full length or over the three regions of CDRs 1, 2 and 3). %, 90%, 92%, 95%, 97%, 98%, 99% or higher identity.
  • the variant of the VH sequence comprises at least one and no more than the reference VH sequence in amino acid sequence (preferably over the full length or over the three regions of CDRs 1, 2 and 3) 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably sequence differences do not occur in the CDR regions.
  • the variant of the VL sequence has at least 80% amino acid sequence compared to the reference VL sequence (preferably over the full length or over the three regions of CDRs 1, 2 and 3) , 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity.
  • the variant of the VL sequence comprises at least one and No more than 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions). Preferably sequence differences do not occur in the CDR regions.
  • the antibody of the invention comprises, or consists of, the heavy chain variable region and light chain variable region VH/VL sequence pair of any of the antibodies listed in Table B.
  • the invention also provides variants of the antibody, eg, variants that are at least 95-99% identical or contain no more than 10 amino acid changes in VH, VL, or VH and VL.
  • the heavy chain variable region of the antibody variant comprises no more than 10, preferably no more than 1, over 1 or more CDR (preferably all 3 CDRs) regions relative to the reference antibody 5 (eg, 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the light chain variable region VL of the antibody variant comprises no more than 10, preferably no more than 10 CDR regions (preferably all 3 CDRs), relative to the reference antibody More than 5 (eg, 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions).
  • the antibody of the invention comprises a heavy chain constant region and/or a light chain constant region.
  • the antibodies of the invention comprise a heavy chain Fc region, eg, an Fc region of an IgGl, IgG2, IgG3 or IgG4 isotype.
  • the antibodies of the invention contain an IgGl-Fc region, especially a human IgGl-Fc region.
  • the antibodies of the invention comprise a kappa light chain constant region, eg, a human kappa light chain constant region.
  • the antibody may comprise a heavy chain constant region with the same or altered effector function relative to the native Fc region.
  • effector functions include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP).
  • effector function occurs when the variable regions of the antibody bind to surface antigens on cells (eg, tumor cells) and the Fc region of the antibody binds to Fc receptors on effector cells (eg, T effector cells).
  • humoral immunity is mostly mediated through the interaction of the antibody Fc region with C1q and the complement cascade.
  • the cellular immune response is mostly mediated by the interaction of the Fc region of the antibody with the Fc ⁇ receptor (ie, Fc ⁇ R).
  • FcyRI, FcyRIIA, FcyRIIIA and FcyRIIIB are activating FcyRs.
  • FcyRIIB is an inhibitory FcyR.
  • Intracellular signaling of activating receptors is mediated by phosphorylation of the receptor's intracellular ITAM motif, which leads to effector functions such as ADCC, ADCP, and inflammatory responses by inducing cytokine release.
  • Cell signaling of the inhibitory receptor Fc ⁇ RIIB is mediated by phosphorylation of the receptor's intracellular ITIM motif, which functions to balance the activating signaling pathway.
  • the interaction of antibodies with Fc ⁇ R and C1q mainly depends on the hinge and CH2 amino acid sequence and the glycosylation of the CH2 region.
  • the antibodies of the invention comprise a heavy chain constant region that binds an activating FcyR.
  • the antibody comprises a heavy chain constant region that binds an FcR selected from the group consisting of FcyRIIIA(CD16a), FcyRIIIA(CD16a) F158 variant, FcyRIIIA(CD16a) V158 variant.
  • the antibody comprises a constant region of human IgGl or IgG3 subclass.
  • the anti-TNFR2 antibodies of the invention induce ADCC in the presence of effector cells such as NK cells.
  • the antibodies of the invention comprise the human IgG1 constant region amino acid sequence of SEQ ID NO:62, or at least one, two or three, but not more than 20 relative to the amino acid sequence of SEQ ID NO:62 , an amino acid sequence with 10 or 5 amino acid changes, or a sequence that is at least 95-99% identical to the amino acid sequence of SEQ ID NO:62.
  • the antibody of the invention comprises a light chain constant region.
  • the light chain constant region is a human kappa light chain constant region.
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO:63, or comprises at least one, two or three, but not more than 20, 10 relative to the amino acid sequence of SEQ ID NO:63 An amino acid sequence that is altered by 1 or 5 amino acids, or a sequence that is at least 95-99% identical to the amino acid sequence of SEQ ID NO:63.
  • the antibody of the invention comprises a heavy chain
  • the heavy chain comprises or comprises at least one, two or three relative to the amino acid sequence selected from the group consisting of SEQ ID NOs: 32-37 and 66, but not more than 20, 10 or 5 amino acid changes in the amino acid sequence, or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical thereto amino acid sequence.
  • the amino acid changes do not occur in the CDR regions, more preferably, in the variable regions.
  • the antibody of the invention comprises a light chain
  • the light chain comprises or comprises at least one, two or three relative to the amino acid sequence selected from SEQ ID NOs: 38-44 and 67, but not more than 20, 10 or 5 amino acid changes in the amino acid sequence, or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical thereto amino acid sequence.
  • the amino acid changes do not occur in the CDR regions, more preferably, in the variable regions.
  • the antibody of the invention comprises a heavy chain sequence and/or a light chain sequence selected from the group consisting of:
  • the variant comprises at least one, two or three, but not more than 20, 10 or 5 amino acid changes in the amino acid sequence compared to the corresponding reference sequence, or has at least 80%, 85%, 90% , 92%, 95%, 97%, 98%, 99% or more identical amino acid sequences.
  • the amino acid changes do not occur in the CDR regions, more preferably in the variable regions.
  • the heavy chain and/or light chain of an anti-TNFR2 antibody or fragment thereof of the invention further comprises a signal peptide sequence.
  • the invention provides antibodies, eg, murine antibodies, chimeric antibodies, and humanized antibodies, that specifically bind TNFR2 (eg, human TNFR2), as isolated and characterized in the Examples.
  • TNFR2 eg, human TNFR2
  • the antibody variable region VH and VL sequences of these exemplary antibodies of the invention are listed in FIG. 16 .
  • Exemplary CDR sequences for antibodies are listed in FIG. 15 .
  • the invention provides variants of any of the antibodies described herein, particularly the exemplary antibodies listed in Table B.
  • the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the pre-altered antibody.
  • the alteration does not result in the antibody variant losing binding to the antigen, but optionally may confer properties such as increased antigen affinity and different effector functions.
  • variable heavy or light chain regions of an antibody, or each CDR region may be altered individually or in combination.
  • the antibody variant has at least 80%, 85%, 90%, or 95% or 99% or more amino acid identity to the reference antibody over a region of the antibody sequence of interest.
  • an antibody of the invention compared to a reference antibody (eg, one of the antibodies listed in Table B), has at least 90%, 91%, 92%, 93%, at least 90%, 91%, 92%, 93%, over the 3 heavy chain CDR regions, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.
  • an antibody of the invention compared to a reference antibody (eg, one of the antibodies listed in Table B), has at least 90%, 91%, 92%, 93%, 94% over the 3 light chain CDR regions , 95%, 96%, 97%, 98%, or 99% or higher identity.
  • the antibodies of the invention, compared to a reference antibody have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity.
  • the antibody of the invention has at least 80%, 85%, 90%, 91%, 92% in the variable region of the heavy chain compared to a reference antibody (eg, one of the antibodies listed in Table B) , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • the antibody of the invention has at least 80%, 85%, 90%, 91%, 92% in the light chain variable region compared to a reference antibody (eg, one of the antibodies listed in Table B) , 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • the antibody of the invention has at least 80%, 85%, 90% in the heavy chain variable region and light chain variable region compared to a reference antibody (eg, one of the antibodies listed in Table B) , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity.
  • a reference antibody eg, one of the antibodies listed in Table B
  • changes can be made to the Fc region of the antibody. Changes to the Fc region can be made alone or in combination with the above-described changes to the framework and/or CDR regions.
  • the Fc region can be altered, eg, to alter one or more functions of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • the antibodies of the invention can also be chemically modified (eg, linked to PEG) or altered in their glycosylation pattern.
  • the antibody may comprise a modified Fc region variant having altered effector function compared to the unmodified parental Fc region.
  • one or more alterations e.g. enhancement or An anti-TNFR2 antibody of the invention that reduces or eliminates
  • effector function such as an anti-TNFR2 antibody having an increased function selected from: increased ADCC, CDC, ADCP, and/or serum half-life relative to the unmodified parental Fc region .
  • the antibody can be chemically modified, eg, by altering the antibody's glycosylation pattern, to alter its functional properties.
  • an antibody Fc region may comprise an Fc-region having one or more amino acid substitutions that enhance ADCC activity, eg, substitutions at positions 298, 333 and/or 334 of the Fc-region (EU numbering of residues ).
  • the Fc-region can also be altered to result in altered (ie, increased or decreased) C1q binding and/or complement-dependent cytotoxicity (CDC) (see, eg, US 6,194,551, WO99/51642 and Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184).
  • the antibody comprises an Fc region variant having increased ADCC activity compared to the native unmodified parent Fc region.
  • Exemplary alternatives include: 236A, 239D, 239E, 268D, 267E, 268
  • Binding sites on human IgG1 to FcyR1, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591 -6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcyRIII.
  • the antibody Fc can be altered to increase or decrease its degree of glycosylation and/or to alter its glycosylation pattern. Addition or deletion of glycosylation sites to Fc can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites. For example, one or more amino acid substitutions can be made to eliminate one or more glycosylation sites, thereby eliminating glycosylation at that site.
  • Antibodies can be prepared with altered types of glycosylation, eg, low or no-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC capacity of antibodies.
  • the present invention also contemplates antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. These antibody variants may have improved CDC function.
  • alterations in the degree and/or pattern of antibody glycosylation can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery.
  • Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies are expressed to produce antibodies with altered glycosylation.
  • the invention also contemplates antibody variants that possess some, but not all, effector functions, making them ideal candidates for certain applications where the in vivo half-life of the antibody is important, but Certain effector functions, such as complement and ADCC, are unnecessary or detrimental.
  • the Fc region may comprise mutations that eliminate or attenuate effector function, such as a human IgGl Fc region with mutations P329G and/or L234A and L235A, or a human IgG4 Fc region with mutations P329G and/or S228P and L235E.
  • cysteine-engineered antibodies eg, "thioMAbs”
  • one or more residues of the antibody are replaced with cysteine residues.
  • the number of cysteine residues in the hinge region of an antibody can be altered, eg, to facilitate light and heavy chain assembly or to increase or decrease the stability of the antibody.
  • Residues are eg US Pat. No. 5,677,425.
  • the antibodies provided herein can be further modified to contain non-proteinaceous moieties.
  • Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), eg, to increase the (eg, serum) half-life of an antibody.
  • PEG polyethylene glycol
  • Methods for protein PEGylation are known in the art and can be applied to the antibodies of the invention. See eg EP 0154 316 and EP 0401384.
  • the present invention provides nucleic acids encoding any of the above anti-TNFR2 antibodies or fragments thereof.
  • a vector comprising the nucleic acid is also provided.
  • the vector is an expression vector.
  • Host cells comprising the nucleic acid or the vector are also provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells).
  • the host cell is prokaryotic.
  • the invention provides nucleic acids encoding any of the above anti-TNFR2 antibodies or fragments thereof.
  • the nucleic acid may comprise nucleic acid encoding the amino acid sequence of the light chain variable region and/or heavy chain variable region of an antibody, or nucleic acid encoding the amino acid sequence of the light chain and/or heavy chain of the antibody.
  • An exemplary nucleic acid sequence encoding an antibody heavy chain variable region comprises at least 80%, 85%, 90%, 91% with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, 55 and 68 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequences, or comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 45, 47, 49, 51, 53, 55 and 68 nucleic acid sequences.
  • Exemplary nucleic acid sequences encoding antibody light chain variable regions include at least 80%, 85%, 90% with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, 54, 56, 57, and 69 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleic acid sequences, or comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 46, 48, 50, 52, Nucleic acid sequences of 54, 56, 57, and 69.
  • the polypeptides encoded by these polynucleotides are capable of exhibiting TNFR2 antigen binding ability when expressed from a suitable expression vector.
  • polynucleotides encoding at least one CDR region and generally all three CDR regions from the heavy chain VH or light chain VL sequences of the TNFR2 binding antibodies described above.
  • the polynucleotide encodes the complete or substantially complete variable region sequence of the heavy and/or light chain of the TNFR2-binding antibody described above.
  • each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences because of codon degeneracy.
  • the nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a heavy chain constant region, such as the constant region sequence shown in SEQ ID NO: 62 or a sequence substantially identical thereto.
  • the nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a light chain constant region sequence, such as the sequence shown in SEQ ID NO: 63 or a sequence substantially identical thereto.
  • TNFR2-binding antibodies or antigen-binding fragments thereof can be obtained by de novo solid phase DNA synthesis, or by PCR mutagenesis, using methods well known in the art.
  • one or more vectors comprising the nucleic acids of the invention are provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • a host cell comprising the vector.
  • Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required.
  • the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or antigen-binding fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which the glycosylation pathway has been "humanized” result in antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
  • Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines engineered for growth in suspension can be used.
  • Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293HEK or 293 cells, eg, Graham et al., J. Gen Virol. 36:59 (1977) described in) etc.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • a method of making an anti-TNFR2 antibody comprises, under conditions suitable for expression of the antibody, culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, and optionally The antibody is recovered from the host cell (or host cell culture medium).
  • nucleic acid encoding the antibody eg, the antibodies described above
  • nucleic acids are readily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains).
  • anti-TNFR2 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • Binding of antibodies to human TNFR2 can be determined by methods known in the art, such as ELISA, Western blotting, etc., or by the exemplary methods disclosed in the Examples herein.
  • assays can be performed using flow cytometry in which the antibody is reacted with a cell line expressing human TNFR2, eg, CHO cells transfected to express human TNFR2 on the cell surface.
  • Other cells are also suitable for flow cytometry, including tumor cells or primary Treg cells expressing human TNFR2.
  • antibody binding including binding kinetics (eg, KD values), can be determined in a bioluminescence interferometry assay using recombinant TNFR2 protein.
  • binding to human and other species-derived TNFR2 equilibrates dissociation constants using biooptical interferometry (eg, Fortebio affinity measurements).
  • the inhibitory/antagonistic antibodies of the present invention against TNFR2 can block the activation of the TNFR2 signaling pathway by the ligand TNF ⁇ by binding to cell membrane TNFR2.
  • the blocking activity relies on the blocking of binding of TNFR2 by an antibody to its natural ligand, TNF ⁇ , such as the example antibodies 3C4 and 69B1 and their humanized forms, or those having the same or similar VH and VL sequences.
  • the blocking activity is independent of the blocking of the binding of TNFR2 by an antibody to its natural ligand, TNF ⁇ , such as the example antibody 72G8 and its humanized forms, or those having the same or similar VH and VL sequences.
  • Antibodies that can directly inhibit the multimerization of TNFR2 on the cell surface, thereby inhibiting TNFR2 receptor activation can directly inhibit the multimerization of TNFR2 on the cell surface, thereby inhibiting TNFR2 receptor activation.
  • the inhibitory effect of the antibodies of the invention on the TNF ⁇ -activated TNFR2 signaling pathway can be detected in Jurkat cells overexpressing TNFR2 using, for example, the Jurkat TNFR2 NF- ⁇ B luciferase reporter assay system.
  • antibodies of the invention are tested for inhibition of TNF ⁇ -activated TNFR2 signaling according to the methods described in Example 8.
  • the ADCC killing effect of the antibody of the present invention on target cells expressing TNFR2 can be detected by the biological activity detection of ADCC reporter gene.
  • the ADCC reporter gene bioactivity assay selects an earlier event in the activation of the ADCC mechanism of action pathway as a readout indicator for the assay: the activation of gene transcription mediated by the NFAT (nuclear factor in activated T cells) pathway in effector cells.
  • ADCC reporter assays use engineered T cells as effector cells (eg, Jurkat cells) that stably express the Fc ⁇ RIIIa receptor (eg, the V158 high-affinity mutant) and firefly fluorescence driven expression by NFAT response elements Vegetase.
  • the biological activity of the antibody in the ADCC mechanism of action is quantified by luciferase produced by NFAT pathway activation, while the luciferase activity in effector cells is quantified by bioluminescence readout.
  • ADCC reporter gene bioactivity assays a good assay response will only be obtained when target cells with the correct surface antigen, the correct specific antibodies, and effector cells expressing Fc ⁇ RIIIa are present simultaneously.
  • the ADCC activity of the antibody of the present invention is detected by the ADCC reporter gene detection method described in Example 14.
  • the present invention also provides assays for identifying biologically active anti-TNFR2 antibodies.
  • Biological activities can include, for example, binding TNFR2 (eg, binding human TNFR2), blocking TNFR2 (eg, binding human TNFR2) binding to TNF ⁇ molecules, inhibiting signaling mediated by TNF ⁇ binding to TNFR2, triggering ADCC activity, and/or inhibiting tumor growth .
  • binding TNFR2 eg, binding human TNFR2
  • any of the above assays can be performed using immunoconjugates or multispecific antibodies of the invention in place of or in addition to anti-TNFR2 antibodies.
  • the present invention provides multispecific (including bispecific) antibody molecules that specifically bind TNFR2, preferably human TNFR2.
  • the antibody of the invention (or antigen-binding fragment thereof) forms a first binding specificity for TNFR2.
  • the multispecific antibody further comprises a second specificity for one of the following, or further comprises second and third binding specificities for both of the following molecules. Said second and third binding specificities can, for example, be directed against another antigen expressed on the surface of tumor cells.
  • the binding specificity is provided by the "binding site” or "antigen binding site” of the antibody (the region of the antibody molecule that actually binds to the antigen).
  • the antigen binding site consists of a VH/VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • a "multispecific" antibody is an antibody having at least two antigen-binding sites, each of which may be associated with a different expression of the same antigen. or bind to different epitopes of different antigens. For multispecific antibodies and their preparation, see eg the descriptions in WO 2009/080251, WO 2009/080252, WO 2009/080253 and WO 2010/145793.
  • the present invention provides immunoconjugates produced by conjugating an antibody of the present invention to a heterologous molecule.
  • an antibody of the invention (or antigen-binding fragment thereof) is conjugated to a therapeutic or diagnostic agent.
  • the antibodies of the invention can be conjugated to heterologous molecules in the form of full-length antibodies or antibody fragments.
  • conjugation is carried out in the form of Fab fragments, Fab' fragments, F(ab)'2 fragments, single chain scFab antibodies, single chain scFv, etc. fragments.
  • Linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers.
  • the linker is a "cleavable linker" that facilitates release of the polypeptide after delivery to the target site.
  • acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al. Cancer Research 52(1992) 127-131; US 5,208,020) .
  • Therapeutic agents suitable for use in the conjugate include, but are not limited to, cytotoxins (eg, cytostatic or cytotoxic agents), drugs, or radioisotopes.
  • cytotoxic agents eg chemotherapeutic agents
  • cytotoxic agents include, but are not limited to: radioisotopes; growth inhibitors; enzymes and fragments thereof such as nucleic acid hydrolases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants; and various known anti-tumor or anti-cancer agents.
  • the antibodies of the invention can be conjugated to a diagnostic or detectable agent.
  • a diagnostic or detectable agent can be used as part of a clinical assay (eg, to determine the efficacy of a particular therapy) for monitoring or predicting the onset, development, progression and/or severity of a disease or disorder.
  • diagnosis and detection can be accomplished by conjugating antibodies to detectable agents including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase; prosthetic groups, such as, but not limited to, streptavidin. Avidin/biotin and avidin/biotin; fluorescent substances; luminescent substances; radioactive substances; and positron emitting and nonradioactive paramagnetic metal ions used in various positron emission imaging procedures.
  • compositions comprising anti-TNFR2 antibodies, or immunoconjugates or multispecific antibodies thereof, and multinuclei encoding anti-TNFR2 antibodies, or immunoconjugates or multispecific antibodies thereof A composition of glucosides.
  • compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.
  • Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk , glycerol, propylene, glycol, water, ethanol, etc.
  • compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.
  • These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.
  • Anti-TNFR2 antibodies, immunoconjugates or multispecific antibodies of the invention of the desired purity can be prepared by mixing with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)) to prepare a pharmaceutical formulation comprising the present invention, preferably in the form of a lyophilized formulation or an aqueous solution.
  • the antibodies of the present invention may be the sole active agent, or may be combined with other therapeutic agents.
  • Therapeutic agents that can be combined with the antibodies of the invention include, but are not limited to, those that have beneficial therapeutic efficacy for the disease and/or disorder to be treated.
  • the active ingredients may be desired for the particular indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other.
  • other pharmaceutical ingredients may provide anticancer activity.
  • the antibodies of the invention are suitably present in pharmaceutical compositions and formulations in combination with the active ingredient in an amount effective for the intended use.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.
  • the invention also provides a combination product comprising an antibody or antigen-binding fragment thereof, bispecific antibody or immunoconjugate of the invention, and one or more other therapeutic agents (eg, chemotherapeutic agents, other antibodies, cytotoxic agents, anti-tumor drugs, etc.). Combinations of the present invention may be used in the methods of treatment of the present invention.
  • the present invention provides a combination product wherein the other therapeutic agent is, for example, a therapeutic agent such as an antibody effective to stimulate an immune response to further enhance, stimulate or upregulate an immune response in a subject.
  • the combination product is used to prevent or treat tumors.
  • the tumor is cancer, eg, colon cancer, chronic myeloid leukemia.
  • the invention provides methods and uses of using the TNFR2 antibodies or antigen-binding fragments thereof of the invention, for example, in vivo and in vitro:
  • the methods and uses of the present invention relate to the treatment of disease in an individual subject.
  • the methods and uses of the invention involve detecting the presence of TNFR2 in a sample from, eg, a subject.
  • the present invention also provides the use of a TNFR2 antibody or antigen-binding fragment thereof of the present invention in the manufacture of a product (eg, a pharmaceutical composition or drug product or assay product) for use as described above.
  • the present invention provides methods and uses for the prevention or treatment of TNFR2-related diseases including, but not limited to, tumors such as colon cancer or chronic myeloid leukemia in an individual or subject using the antibodies or antigen-binding fragments thereof of the invention.
  • the antibodies of the invention may be administered as the sole active agent, or may be administered in combination with other therapies or therapeutic agents.
  • Such other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells, and destroy tumors by binding and/or blocking these molecules; drugs that activate a subject's immune system, causing it to spontaneously destroy tumors .
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and large mouse.
  • rodents eg, mice and large mouse.
  • the subject is a human.
  • treatment includes 1) therapeutic measures that cure, slow, alleviate the symptoms and/or halt the progression of a diagnosed pathological condition or disorder; and 2) prophylactically or preventive measures, which prevent and/or slow the development of a pathological condition or disorder.
  • subjects in need of treatment include those already suffering from the disorder, those susceptible to the disorder, and those in which the disorder is to be prevented.
  • the individual would benefit from the therapeutic or prophylactic measures and exhibit an increase in the occurrence, recurrence or progression of the disease, disorder, condition, and/or symptom as compared to an individual not receiving the treatment alleviate or improve.
  • the invention relates to the treatment of a disease or disorder; in other embodiments, the invention relates to the prevention of a disease or disorder.
  • treatment of a disease or disorder refers to amelioration of the disease or disorder (ie, slowing or arresting or reducing the progression of the disease or at least one clinical symptom thereof).
  • treating refers to alleviating or ameliorating at least one physical parameter, including those physiological parameters that may not be discernible by the patient.
  • treating refers to modulating a disease or disorder physically (eg, stabilizing a discernible symptom), physiologically (eg, stabilizing a physical parameter), or both.
  • methods for assessing the therapeutic and/or prophylactic effect of a disease or disorder are generally known in the art.
  • treatment of a disease or disorder refers to "prevention" of a disease or disorder, including inhibition of the occurrence or progression of a disease or disorder or symptoms of a particular disease or disorder.
  • the subject is a candidate for a prophylactic regimen.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of a disease or disorder, particularly in subjects at risk for the disease.
  • therapeutically effective amount refers to an anti-TNFR2 antibody or antigen-binding fragment thereof of the invention when administered to a cell, tissue or subject alone or in combination with other therapeutic agents, An amount effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the development of such diseases or conditions.
  • a therapeutically effective amount also refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • a therapeutically effective amount refers only to the amount of that ingredient.
  • a therapeutically effective amount refers to the combined amount of active ingredients that elicits the therapeutic effect, whether administered concurrently, sequentially or simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in the diagnostic criterion or parameter by at least 10%, usually by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.
  • the present invention relates to a method of inhibiting a tumor in a subject, the method comprising administering to the subject an effective amount of any anti-TNFR2 antibody or fragment thereof described herein, or comprising the an immunoconjugate, a multispecific antibody, or a pharmaceutical composition of the antibody or fragment.
  • the present invention also relates to a method of enhancing an anti-tumor immune response of a body in a subject, the method comprising administering to the subject an effective amount of any of the anti-TNFR2 antibodies described herein, or Fragments, or immunoconjugates, multispecific antibodies, or pharmaceutical compositions comprising said antibodies or fragments.
  • administration of an anti-TNFR2 antibody or antigen-binding portion thereof of the invention to a tumor-bearing subject kills a TNFR2-bearing tumor. In some embodiments, administration of an anti-TNFR2 antibody or antigen-binding portion thereof of the invention to a tumor-bearing subject stimulates an anti-tumor immune response.
  • the present invention provides a method of treating a tumor, eg, cancer, in a subject, the method comprising administering to the subject an effective amount of any anti-TNFR2 antibody or fragment thereof described herein, or comprising the an immunoconjugate, a multispecific antibody, or a pharmaceutical composition of the antibody or fragment.
  • Cancer can be in early, intermediate or advanced stages or metastatic.
  • Tumors that can be treated with the methods of the present invention include, but are not limited to, colon cancer and chronic myeloid leukemia, for example.
  • the antibodies of the present invention can inhibit tumor growth by targeting tumor cells and/or Treg cells that express TNFR2 on the cell surface.
  • the antibodies of the invention can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal, Also, intralesional administration if required for local treatment.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • administration may be by any suitable route, eg, by injection, eg, intravenously or subcutaneously.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • an antibody of the invention when used alone or in combination with one or more other therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease , whether the antibody is administered for prophylactic or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.
  • the antibody is suitably administered to the patient in a single treatment or over a series of treatments.
  • compositions, multispecific antibodies or immunoconjugates of the present invention may be administered in place of the antibodies or antigen-binding portions of the present invention.
  • a composition, multispecific antibody or immunoconjugate of the present invention may be further administered.
  • the invention also provides the use of an anti-TNFR2 antibody, composition, immunoconjugate, multispecific antibody of the invention in the manufacture of a medicament for use in the aforementioned methods (eg, in therapy).
  • the present invention also provides methods and kits for detecting TNFR2 in a sample, wherein the method comprises: (a) contacting the sample with an antibody or antigen-binding fragment or immunoconjugate of the present invention; and (b) detecting the Formation of a complex between an antibody or antigen-binding fragment or immunoconjugate and the TNFR2 protein.
  • the sample is from a cancer patient, eg, a skin cancer patient.
  • the detection can be in vitro or in vivo.
  • the term "detection” includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (eg, FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg RT-PCR).
  • the biological sample is blood, serum, or other fluid sample of biological origin.
  • the biological sample comprises cells or tissues.
  • the biological sample is from a hyperproliferative or cancerous lesion.
  • the TNFR2 to be detected is human TNFR2.
  • an anti-TNFR2 antibody is used to select subjects suitable for treatment with an anti-TNFR2 antibody, eg, wherein TNFR2 is a biomarker for selection of said subjects.
  • the antibodies of the invention can be used to diagnose cancers or tumors, eg, to evaluate (eg, monitor) a subject for treatment or progression of a disease described herein (eg, a hyperproliferative or cancerous disease), its diagnosis, and/or Staging.
  • labeled anti-TNFR2 antibodies include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.
  • Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H and 131I, fluorophores such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbrella umbelliferone, luceriferase, eg, firefly luciferase and bacterial luciferase (US Patent No.
  • luciferin 2,3-dihydrophthalazine dione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes utilizing hydrogen peroxide dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/avidin , spin tags, phage tags, stabilized free radicals, and more.
  • HR horseradish peroxidase
  • alkaline phosphatase beta-galactosidase
  • glucoamylase lysozyme
  • carbohydrate oxidase e.g., glucose oxid
  • mice were immunized with human TNFR2 protein, and then the spleen cells of the mice were fused with myeloma cells to obtain hybridoma cells capable of expressing positive antibodies.
  • the spleen was taken from the immunized animal to prepare a spleen cell suspension. After filtration and lysis of red blood cells, splenocytes were suspended in 20 ml of basal medium and counted.
  • SP2/0 cells (ATCC, CRL-1581) were resuspended in 20 ml of basal medium and counted. SP2/0 and splenocytes were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 min. After removal of the supernatant, the mixed cells were resuspended in 10 ml of fusion buffer (BTXpress, 47-0001). Add 15 ml of fusion buffer, centrifuge at 1000 rpm for 5 min, and remove the supernatant. After repeating the above steps, resuspend the cells with an appropriate amount of fusion buffer, and adjust the mixed cell density to 1 ⁇ 10 7 cells/ml.
  • the parameters of the electrofusion apparatus are set as follows. Add 2 ml of cell suspension to each electrofusion dish for electrofusion.
  • the cells were placed in the electrofusion dish for 5 min at room temperature. Transfer cells into centrifuge tubes and dilute cells to 1-2 x 104 cells/ml with selection medium (table below). Add 100 ⁇ l of cell suspension to each well of a 96-well plate. The selection medium was changed on day 7 after fusion. Screening was performed after day 10 of culture (or longer, depending on cell growth status).
  • the hybridoma cells expressing specific anti-hTNFR2 antibody were screened by Elisa assay.
  • the antibodies expressed by the screened hybridoma cells are sequenced by PCR, the variable region genes of the antibodies are determined, and the chimeric antibody is screened, humanized, and affinity matured to obtain candidate sequences at different stages.
  • the 3 antibodies (3C4, 69B1, 72G8) exemplified by the present invention and their humanized versions of CDR regions, light chain variable regions and heavy chain variable regions, amino acid sequences of light and heavy chains, and corresponding nucleosides The acid sequences are listed in the table below.
  • Table 1 SEQ ID NO: numbering of each antibody-related sequence involved in the present invention
  • HC is formed by fusing the VH sequence to the IgG1 portion of SEQ ID NO:62
  • LC is formed by fusing the VL sequence to the C ⁇ chain of SEQ ID NO:63.
  • Antibody-expressing CHO-S cell lines were generated using the GS Xceed TM Gene Expression System kit (Lonza) according to the manufacturer's instructions. The DNA sequences of the heavy and light chains of the antibody molecule were first inserted into the same pCHO1.0 plasmid, with the heavy chain upstream of the light chain. Then, the constructed pCHO1.0 plasmid was transferred into CHO cell line by chemical transfection and electrotransfection. After 48 hours of transfection, ForteBio was used to detect the antibody production to judge the transfection efficiency. The transfected cells were subjected to two rounds of pressurized screening to obtain a pool of cells with high expression of antibodies. After that, the cell pool was expanded, the antibody was expressed in large quantities, and the cell supernatant was collected and purified with Protein A, so that the purity of the antibody was >95%.
  • the vector pcDNA3.1 was used for transient expression of antibodies in HEK293 cells.
  • the cDNAs encoding the heavy and light chains of the antibody were first cloned into the pcDNA3.1 vector.
  • the vectors with heavy and light chains of antibody molecules were transferred into HEK293 cells by chemical transfection.
  • the chemical transfection reagent used was PEI (purchased from Polysciences), and cultured 293HEK (Invitrogen) was transiently transfected according to the protocol provided by the manufacturer. After transfection, the medium was discarded and cells were diluted to 4 x 106/ml with fresh EXPI293 medium (Gibco). Cells were cultured for 7 days at 37°C, 5% CO2, with fresh medium fed every 48 hours. After 7 days, centrifuge at 1300 rpm for 20 min. Take the supernatant and purify the supernatant with Protein A to make the antibody purity >95%.
  • Example 3 Determination of the binding kinetics of the chimeric antibody of the present invention and the antigen by the biofilm thin-layer interference technology
  • the present invention uses a bio-optical interferometry (ForteBio) assay to determine the balance of the above-mentioned three exemplary chimeric antibodies of the present invention binding to human TNFR2 (hTNFR2), cynomolgus monkey TNFR2 (cTNFR2), murine TNFR2 (mTNFR2), and human TNFR1 (hTNFR1).
  • Dissociation constant (K D ) As a comparison, the equilibrium dissociation constant (K D ) of the positive control antibody OPI (humanized antibody SBT-002e in WO2017083525, VH sequence SEQ ID NO:58, VL sequence SEQ ID NO:59) was determined.
  • ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p.270-8). Briefly, the sensor was equilibrated off-line in assay buffer for 30 minutes, then detected on-line for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded on-line onto the AHQ sensor (ForteBio) for ForteBio affinity measurements. The sensor with the loaded antibody was then exposed to 100 nM of hTNFR2, cTNFR2, mTNFR2, hTNFR1 antigen for 5 minutes, after which the sensor was transferred to assay buffer for dissociation for 5 minutes for dissociation rate measurement. Kinetic analysis was performed using a 1:1 binding model.
  • Example 4 Binding experiment of hybridoma antibody and CHO-S cells overexpressing human TNFR2 (CHOS-hTNFR2) or CHO-S cells overexpressing cynomolgus monkey TNFR2 (CHOS-cTNFR2)
  • the cDNAs encoding human TNFR2 (SEQ ID NO: 60, NM_001066.2) and cynomolgus monkey TNFR2 (SEQ ID NO: 61, XP_005544817.1) were cloned into pCHO1.0 vector (Invitrogen) and transfected into CHO-S cells (Invitrogen), CHO-S cells overexpressing human TNFR2 (CHOS-hTNFR2) and CHO-S cells overexpressing cynomolgus TNFR2 (CHOS-cTNFR2) were generated.
  • CHOS-hTNFR2 cells or CHOS-cTNFR2 cells were counted and diluted to 2 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate.
  • the cell culture medium was removed by centrifugation at 300 g for 5 min.
  • Dilutions of samples (hybridoma antibodies 3C4, 69B1, 72G8 and positive control antibody OPI, respectively) (antibody dilution method: the highest antibody concentration is 300nM, two-fold serial dilution in PBS) were added to the U-plate and the cells were resuspended, 100 ⁇ l/well, let stand on ice for 30 min.
  • the supernatant was removed by centrifugation at 400 g for 5 min, and the cells were washed twice with PBS.
  • PBS was removed by centrifugation at 300 g for 5 min, 100 ⁇ l of anti-mouse IgG Alexa Fluor-488-labeled secondary antibody (Jackson ImmunoResearch; 115-545-072) (1:100 dilution in PBS) was added to each well, and incubated on ice for 30 min in the dark.
  • the supernatant was removed by centrifugation at 400 g for 5 min, and the cells were washed three times with PBS. Cells were resuspended in 200 ⁇ l 1 ⁇ PBS and detected by FACS.
  • hybridoma antibodies 3C4, 69B1, 72G8 and control antibody OPI to block the binding of human TNF ⁇ to hTNFR2 was detected by ELISA.
  • the hTNFR2 protein was resuspended in PBS and dissolved to a concentration of 2ug/ml, and coated onto an ELISA plate overnight. After blocking with 5% BSA for 1 h, the biotin antigen Recombinant Biotinylated TNF ⁇ protein (ACRO) was diluted to 3 ug/ml, 20 ⁇ l/well.
  • the antibodies (3C4, 69B1, 72G8) prepared as described above were serially diluted starting from the highest concentration of 300 nM, with a total of 8 or 12 dilution gradients, 200 ⁇ l/well, and incubated on ice in PBS for 30 min.
  • the antigen-antibody mixture was incubated to the ELISA plate for 90 min, washed three times with PBS, discarded the supernatant, added 100 ⁇ l Avidin-HRP (Invitrogen)/well diluted 1:5000, and washed six times with PBS for 30 min at room temperature. Use 100ul/well of TMB chromogenic solution (solarbio) to develop color for 1 min, and stop with 100ul/well of stop solution (solarbio).
  • the microplate reader reads OD450 and OD620 .
  • Example 6 Binding experiment of chimeric antibody with CHO-S cells overexpressing human TNFR2 (CHOS-hTNFR2) or CHO-S cells overexpressing cynomolgus TNFR2 (CHOS-cTNFR2)
  • the cDNAs encoding human TNFR2 (SEQ ID NO: 60) and cynomolgus monkey TNFR2 (SEQ ID NO: 61) were cloned into pCHO1.0 vector (Invitrogen) and transfected into CHO-S cells (Invitrogen) to produce CHO-S cells overexpressing human TNFR2 (CHOS-hTNFR2) and CHO-S cells overexpressing cynomolgus TNFR2 (CHOS-cTNFR2).
  • CHOS-hTNFR2 cells or CHOS-cTNFR2 cells were counted and diluted to 2 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate.
  • the cell culture medium was removed by centrifugation at 300 g for 5 min.
  • Dilutions of samples chimeric antibody 3C4, 69B1, 72G8 and positive control antibody OPI, respectively
  • antibody dilution method the highest antibody concentration is 300nM, three-fold serial dilution in PBS
  • the cells are resuspended , 100 ⁇ l/well, and let stand on ice for 30 min.
  • the supernatant was removed by centrifugation at 400 g for 5 min, and the cells were washed twice with PBS.
  • PBS was removed by centrifugation at 300 g for 5 min, 100 ⁇ l of anti-human Fc PE-labeled secondary antibody (SoutherBiotech; 2040-09) (1:100 diluted in PBS) was added to each well, and incubated on ice for 30 min in the dark.
  • the supernatant was removed by centrifugation at 400 g for 5 min, and the cells were washed three times with PBS. Cells were resuspended in 200 ⁇ l of 1 ⁇ PBS and detected by FACS.
  • the hTNFR2 protein was resuspended in PBS and dissolved to a concentration of 2ug/ml, and coated onto an ELISA plate overnight. After blocking with 5% BSA for 1 h, the biotin antigen Recombinant Biotinylated TNF ⁇ protein (ACRO) was diluted to 2ug/ml, 10 ⁇ l/well.
  • the antibodies (3C4, 69B1, 72G8) prepared as described above were serially diluted starting from the highest concentration of 300 nM, with a total of 8 or 12 dilution gradients, 100 ⁇ l/well, and incubated on PBS ice for 30 min.
  • the antigen-antibody mixture was incubated to the ELISA plate for 90 min, washed three times with PBS, discarded the supernatant, added 100 ⁇ l Avidin-HRP (Invitrogen)/well diluted 1:5000, and washed six times with PBS for 30 min at room temperature.
  • Example 8 Chimeric antibody inhibits TNFR2-mediated activation of NF- ⁇ B signaling pathway
  • Inhibitory antibodies against TNFR2 can activate the TNFR2 signaling pathway by binding to the cell membrane TNFR2 to block the ligand TNF ⁇ .
  • the Jurkat TNFR2 NF- ⁇ B luciferase reporter system was used to verify the inhibitory effect of the antibody of the present invention on the TNFR2 signaling pathway activated by TNF ⁇ in Jurkat cells overexpressing TNFR2.
  • Jurkat TNFR2 NF- ⁇ B reporter cells were added to each well at 5*10 4 cell/well, and the cells were incubated at a final concentration of 50ng TNF ⁇ and different concentrations of antibody (the highest concentration was 20nM, followed by 1:2 equal ratio dilution), incubate in a 37°C CO2 incubator for 6h, add 100ul luciferase substrate solution to each well and shake for 2min. Multifunctional microplate reader (Molecular Device i3) reading. Calculate the fold change in antibody fluorescence readings according to the following formula:
  • Fig. 7 The experimental results (Fig. 7) showed that both chimeric antibodies 3C4 and 69B1, which blocked the binding of TNFR2 to TNF ⁇ , could inhibit the activation of TNFR2 NF- ⁇ B signaling pathway induced by TNF ⁇ , and the inhibitory activity of the 3C4 and 69B1 antibodies was better than that of the positive control antibody OPI.
  • the 72G8 antibody exhibits a novel mechanism of inhibition of TNFR2 functional activity, which can directly inhibit TNFR2 multimerization without blocking TNFR2 binding to TNF ⁇ , thereby inhibiting TNF ⁇ -induced TNFR2NF- ⁇ B signaling pathway activation.
  • the cDNA encoding human TNFR2 was cloned into pCHO1.0 vector (Invitrogen) and transfected into CHO-S cells (Invitrogen) to generate CHO-S cells overexpressing human TNFR2 (CHOS-hTNFR2).
  • CHOS-hTNFR2 cells were counted and diluted to 2 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate. The cell culture medium was removed by centrifugation at 300 g for 5 min.
  • the samples (respectively humanized antibodies hz3C4.7, hz69B1.5, hz69B1.6, hz69B1.11, hz69B1.20, chimeric antibody 69B1, and positive control antibody OPI) (antibody dilution method: the highest antibody concentration is 300nM, three-fold dilution in PBS) was added to the U-plate and resuspended the cells, 100 ⁇ l/well, and let stand on ice for 30min.
  • the supernatant was removed by centrifugation at 400 g for 5 min, and the cells were washed twice with PBS.
  • PBS was removed by centrifugation at 300 g for 5 min, 100 ⁇ l of anti-human Fc PE-labeled secondary antibody (SoutherBiotech; 2040-09) (1:100 diluted in PBS) was added to each well, and incubated on ice for 30 min in the dark.
  • the supernatant was removed by centrifugation at 400 g for 5 min, and the cells were washed three times with PBS. Cells were resuspended in 200 ⁇ l 1 ⁇ PBS and detected by FACS.
  • Example 7 In a similar manner to Example 7, the humanized antibodies hz3C4.7, hz69B1.5, hz69B1.6, hz69B1.11, hz69B1.20 and the control antibody OPI were tested for their ability to block the binding of human TNF ⁇ to hTNFR2 by ELISA.
  • Example 12 Humanized antibody inhibits TNFR2-mediated activation of NF- ⁇ B signaling pathway
  • Example 8 In a similar manner to Example 8, the inhibition of TNF ⁇ -activated TNFR2 signaling by the antibodies of the humanized forms of 3C4 and 69B1 was examined in Jurkat cells overexpressing TNFR2 by the Jurkat TNFR2 NF- ⁇ B luciferase reporter system. The results are shown in Figures 10A and 10B.
  • Detection steps centrifuge at 400 g for 5 min, remove the cell culture medium, resuspend K562 or Treg cells in PBS, and after counting, adjust the cell density to 2 ⁇ 10 6 cells/ml, and add 100 ⁇ l/well to a U-bottom 96-well plate. Add the antibody to be tested, make a three-fold serial dilution, and let stand on ice for 30 min. Remove the supernatant at 300 g for 5 min, and wash the cells once with PBS. 300 g, 5 min to remove PBS, and 100 ⁇ l of 1:200 diluted PE-anti-human Fc antibody (SOUTHERN BIOTECH, 2040-09) was added to each well. Incubate on ice for 30 min in the dark. Remove the supernatant at 400 g for 5 min, and wash the cells twice with PBS. Cells were resuspended in 100 ⁇ l PBS and detected by flow cytometry (BD, ACCURIC6 plus).
  • the Jurkat ADCC reporter assay (Promega, G7102) was used to detect the ADCC killing effect of serially diluted humanized antibodies of the present invention on CHOS-hTNFR2 cells.
  • Detection step According to the experimental protocol provided by the supplier, the antibody was serially diluted, the initial concentration was 50nM, and the 1:2 equal ratio was diluted. After that, 2.5 ⁇ 10 4 CHOS-hTNFR2 cells, and 1.75 ⁇ 10 5 Jurkat effector cells were added per well. Incubate in a CO2 incubator at 37°C for 20h, and add luciferase substrate. After 2 min, use a multifunctional microplate reader (MD, I3 multifunctional microplate reader) to detect.
  • MD multifunctional microplate reader
  • K562 cells were used to inoculate NOD/SCID mice to determine the anti-tumor effect of the anti-TNFR2 antibody of the present invention.
  • mice Female NOD/SCID mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the grade was SPF. The quality inspection unit was Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the certificate number was NO.11400700380384. Mice were acclimated for 7 days after arrival before the study began.
  • K562 cells are human chronic myeloid leukemia cells, purchased from ATCC (CAT#: CCL-243), and routinely subcultured in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in Matrigel and adjusted to a cell density of 5 x 106 cells/ml. 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal area of NOD/SCID mice to establish a K562 tumor-bearing mouse model.
  • mice with tumor volume ranging from 25mm 3 to 118mm 3 were selected and randomly divided into groups (8 mice in each group), with an average tumor volume of 65mm 3 in each group. about.
  • the dosage and mode of administration are shown in Table 3.
  • h-IgG purchased from EQUITECH-BIO
  • the mice were administered on the 6th, 10th, 13th, and 17th days after inoculation, twice a week, for a total of four times, and the tumor volume and body weight of the mice were monitored.
  • TGI% 100%*(control group tumor volume – treatment group tumor volume)/( Control group tumor volume – control group pre-dose tumor volume).
  • MC38-TNFR2 cells mouse colon cancer cells expressing human TNFR2 were used to inoculate C57BL/6 mice to determine the anti-tumor effect of the anti-TNFR2 antibody of the present invention.
  • the female C57BL/6 background mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., with a grade of SPF, and the quality inspection unit was Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the certificate number was NO.1100111911055796. Mice were acclimated for 7 days after arrival before the study began.
  • Mouse MC38-TNFR2 cells were independently constructed and routinely subcultured in strict accordance with the instructions for subsequent in vivo experiments. Cells were collected by centrifugation, resuspended in PBS and adjusted to a cell density of 2 x 106 cells/ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of human C57BL/6 mice to establish the MC38-TNFR2 tumor-bearing mouse model.
  • TGI% 100% * (control group tumor volume - treatment group tumor volume)/(control group tumor volume - control group tumor volume before administration ).

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Abstract

提供了TNFR2抗体、其制备方法、组合物和用途。还提供了用于治疗TNFR2相关疾病和/或病症，例如癌症的方法。

Description

抗TNFR2抗体及其用途 技术领域
本发明涉及抗体TNFR2抗体、其制备方法、组合物和用途。本发明还提供了用于治疗TNFR2相关疾病和/或病症，例如癌症的方法。
背景技术
II型肿瘤坏死因子受体(TNFR2，也称作CD120b或p75或TNFRSF1B)，是肿瘤坏死因子(TNF)受体超家族成员。TNFR2作为细胞因子TNFα的受体，在多种类型的癌细胞、和可浸润到肿瘤中抑制免疫系统活性的调节性T细胞(Treg)表面上高表达。TNFα通过与TNFR2结合，可以激活下游NFkB信号传导，导致增强的Treg细胞增殖。已经在各种人和鼠癌症中发现，在肿瘤微环境中存在高丰度的TNFR2-阳性Treg细胞。因此，通过靶向肿瘤微环境的Treg细胞和肿瘤细胞上的TNRF2来治疗癌症，是一种有希望的治疗方案。例如，WO2017/083525公开了一些拮抗性TNFR2抗体并提出其在癌症治疗中用途。
鉴于TNFR2在肿瘤免疫反应中的潜在靶点作用，本领域存在着开发新的TNFR2抗体的需求，尤其是具有强阻断作用和直接的肿瘤杀伤活性的抗TNFR2抗体，以用于疾病治疗，尤其是癌症治疗。
发明概述
本发明人通过深入研究提供了新的抗TNFR2抗体。本发明的抗TNFR2抗体具有强的TNFR2阻断活性，并且部分分子具有新的阻断机制，能够在不阻断TNFα与受体TNFR2结合的情况下直接抑制TNFR2多聚化，从而抑制TNFα/TNFR2信号通路的激活。本发明的抗TNFR2抗体在动物实验中展示了良好的抗肿瘤药效结果。
因此，在一方面，本发明提供了抗TNFR2抗体或其抗原结合片段，其包含：SEQ ID NO:19,21,23,25,27,29,64所示的重链可变区之一的HCDR1、2和3序列，和/或SEQ ID NO:20,22,24,26,28,30,31,65所示的轻链可变区的LCDR1、2和3序列，或所述CDR序列组合的变体。
在一些实施方案中，本发明的抗TNFR2抗体或其抗原结合片段具有以下一个或多个特性：
(i)以高亲和力与人TNFR-2结合，但不结合人TNFR-1；
(ii)具有与猴TNFR-2的交叉免疫反应性；
(iii)与细胞表面上的TNFR-2有效结合，所述细胞尤其是肿瘤细胞例如淋巴瘤细胞和Treg细胞；
(iv)阻断TNFR-2与配体TNFα结合；
(v)抑制由TNFα结合TNFR-2介导的NF-κB信号通路激活；
(vi)激活ADCC信号通路；
(vi)具有抗肿瘤活性，例如抑制肿瘤的生长。
在一些实施方案中，本发明也提供抗TNFR2抗体或其抗原结合片段，所述抗体与本发明示例性抗体(如具有下表B所列抗体VH和VL序列组合的抗体)结合相同或重叠的表位和/或竞争结合TNFR2、和/或抑制(例如，竞争性抑制)本发明示例性抗体。
在一些实施方案中，本发明提供了编码本发明抗体或其抗原结合片段的核酸，包含所述核酸的载体，包含所述载体的宿主细胞。
在一些实施方案中，本发明提供了制备本发明抗体或其抗原结合片段的方法。
在一些实施方案中，本发明提供了包含本发明抗体的免疫缀合物、药物组合物和组合产品。
再一方面，本发明提供应用本发明TNFR2抗体或其抗原结合片段的体内和体外方法和用途，包括例如，但不限于，用于在体内和体外阻断和/或拮抗TNFR-2与其配体TNFα结合、和/或阻断和/或拮抗由所述结合引起的生物学活性，例如TNFR2受体的激活和/或Treg细胞的增殖；和/或用于在体内和体外杀伤表面表达TNFR2的肿瘤细胞。
再一方面，本发明还提供应用本发明抗体或其抗原结合片段预防或治疗TNFR2相关疾病的方法和用途，所述疾病包括但不限于，肿瘤，例如结肠癌或慢性髓系白血病在一些实施方案中，本发明的抗体可以作为唯一活性剂，或可以与其它疗法或治疗剂联合施用。
再一方面，本发明还提供了应用本发明抗体或其抗原结合片段在样品中检测TNFR2的方法和试剂盒。
在下面的附图和具体实施方案中进一步说明本发明。然而，这些附图和具体实施方案不应被认为限制本发明的范围，并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。
附图说明
图1显示流式细胞术测定杂交瘤抗体与人TNFR2过表达稳转细胞株hTNFR2 CHO-S的结合能力。
图2显示流式细胞术测定杂交瘤抗体与食蟹猴TNFR2稳转细胞株cTNFR2 CHO-S的结合能力。
图3显示ELISA法检测杂交瘤抗体对TNFα/TNFR2的阻断作用。
图4显示流式细胞术测定嵌合抗体与人TNFR2过表达稳转细胞株hTNFR2 CHO-S的结合能力。
图5显示流式细胞术测定嵌合抗体与过表达食蟹猴TNFR2稳转细胞株cTNFR2 CHO-S的结合能力。
图6显示ELISA法检测嵌合抗体对TNFα/TNFR2的阻断作用。
图7显示使用Jurkat TNFR2 NF-κB萤光素酶报告基因检测法，检测嵌合抗体对TNFα激活TNFR2信号通路的抑制作用。
图8显示流式细胞术测定人源化抗体与人hTNFR2 CHO-S稳转细胞株的结合能力。
图9显示ELISA法检测人源化抗体对TNFα/TNFR2的阻断作用。
图10显示使用Jurkat TNFR2 NF-κB萤光素酶报告基因检测法，检测人源化抗体对TNFα激活TNFR2信号通路的抑制作用。
图11显示流式细胞术测定人源化抗体与人K562 CML细胞株(A)和原代Treg细胞(B)的结合能力。
图12显示使用Jurkat ADCC萤光素酶报告基因检测法，检测人源化抗体对ADCC信号通路的激活作用。
图13显示K562 matrigel NOD/SCID动物模型检测TNFR2抗体抗肿瘤效应。
图14显示MC38-TNFR2皮下接种C57动物模型检测TNFR2抗体抗肿瘤效应。
图15显示本发明示例性嵌合抗体与人源化抗体的CDR序列。
图16显示本发明示例性嵌合抗体与人源化抗体的VH和VL序列。
发明详述
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或多项的组合。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
在本文中，术语“抗体”是指至少包含轻链或重链免疫球蛋白可变区的多肽，所述免疫球蛋白可变区特异性识别并结合抗原。该术语涵盖各种抗体结构，包括、但不限于单克隆抗体、多克隆抗体、单链抗体或多链抗体、单特异性或多特异性抗体(例如双特异性抗体)、全人源抗体或嵌合抗体或人源化抗体、全长抗体和抗体片段，只要它们呈现期望的抗原结合活性即可。
本领域技术人员明了，“全抗体”(在本文中可与“全长抗体”、“完全抗体”和“完整抗体”互 换使用)包含至少两条重链(H)和两条轻链(L)。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。可变区是抗体的重链或轻链中参与抗体与其抗原结合的结构域。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。抗体的轻链可以基于其恒定结构域的氨基酸序列归入两种类型(称为kappa(κ)和lambda(λ))中的一种。抗体的重链可以取决于其重链恒定区的氨基酸序列而划分为主要5种不同的类型：IgA、IgD、IgE、IgG和IgM，并且这些类型中的几种可以进一步划分成亚类，如，IgG1、IgG2、IgG3和IgG4、IgA1以及IgA2。对应于不同抗体类型的重链恒定区分别称作α、δ、ε、γ和μ。术语“同种型”是指由抗体重链恒定区确定的抗体类型。参见例如Fundamental Immunology，Ch.7(Paul，W.编辑，第二版，Raven Press，N.Y.(1989))(其为所有目的以其整体在此引作参考)。
术语抗体的“抗原结合部分”(在本文中可与“抗体片段”和“抗原结合片段”互换使用)，是指并非完整抗体的分子，其包含完整抗体中用于结合该完整抗体所结合的抗原的部分。如本领域技术人员理解的，抗体的抗原结合部分通常包含来自“互补决定区”或“CDR”的氨基酸残基。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、scFab、Fab’、F(ab’) 2、Fab’-SH、Fv、单链Fv、双链抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)、微抗体(minibody)、单结构域抗体(sdAb)。关于抗体片段的更详细的描述，可以参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993)；邵荣光等人(编辑)，抗体药物研究与应用，人民卫生出版社(2013)；Hollinger等人，PNAS USA 90:6444-6448(1993)；Hudson等人，Nat.Med.9:129-134(2003)。
术语“嵌合抗体”是指可变区序列源自一物种、恒定区序列源自另一物种的抗体，例如，其中可变区序列源自小鼠抗体、恒定区序列源自人抗体的抗体。
术语“人源化抗体”是指将源自其他哺乳动物物种例如小鼠种系的CDR序列接到人构架序列上的抗体。可以在人构架序列内进行额外的构架区修饰，和/或在CDR序列中进行额外的氨基酸修饰，例如以进行抗体的亲和力成熟。
“分离的”抗体是已经与它的天然环境中的组分分离的抗体。在一些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)确定。关于评价抗体纯度的方法的综述，参见，例如，Flatman，S.等，J.Chrom.B 848(2007)79-87。
术语“表位”是指抗体所结合的抗原区域。表位可以由连续的氨基酸形成或者通过蛋白的三级折叠而并置的非连续氨基酸形成。
在本文中，TNRF2是指"肿瘤坏死因子受体2"，也称作TNFRSF1B和CD120b。该受体 是肿瘤坏死因子受体超家族(TNFRSF)的成员，作为细胞表面膜结合受体，可以在癌细胞和肿瘤浸润性Treg细胞上表达。通过调节NF-KB信号通路，TNFR2能够调节促进细胞存活和增殖的基因的转录。在本文中，该表述涵盖TNFR2及其变体、同种型、同源物、和物种同源物。在根据本发明的一个实施方案中，TNFR2是来源于人的TNFR2，例如，SEQ ID NO:60所示的人TNFR2、或与SEQ ID NO:60具有至少95％，甚至至少96％,97％,98％,或99％氨基酸序列同一性的人TNFR2。在根据本发明的另一实施方案中，TNFR2是来源于猴的TNFR2，例如，SEQ ID NO:61所示的人TNFR2、或与SEQ ID NO:61具有至少95％，甚至至少96％,97％,98％,或99％氨基酸序列同一性的人TNFR2。在本文中，TNFR2也可包括TNFR2的片段，诸如包含胞外结构域的片段，例如保持与本发明任何抗体结合能力的片段。在本文中，TNFα是指TNFR2的天然激动剂配体“肿瘤坏死因子α”。在本文中，TNFR1是指"肿瘤坏死因子受体1"，例如UniProtKB登录号P19438下的人TNFR1。
术语“特异性结合”表示抗体选择性地或优先地结合抗原。如果在生物光干涉测量中，抗体以大约5x 10 -7M或更低、大约1x 10 -7M或更低、大约5x 10 -8M或更低、大约1x 10 -8M或更低、大约5x 10 -9M或更低的K D，与人TNFR2结合，则该抗体是“与人TNFR2特异性结合”的抗体。然而，特异性结合人TNFR2的抗体可以与来自其它物种的TNFR2蛋白具有交叉反应性。例如，特异于人TNFR2的抗体，在一些实施方案中，可以与非人物种的TNFR2蛋白交叉反应。在另一些实施方案中，特异于人TNFR2的抗体可以完全特异于人TNFR2而不表现出物种或其它类型的交叉反应性，或仅表现出对某些物种的TNFR2的交叉反应性。
如本文所用的，术语“交叉反应”是指抗体结合来自不同物种的TNFR2的能力。例如，本文所述的结合人TNFR2的抗体还可结合来自其它物种的TNFR2(例如，食蟹猴TNFR2)。测定交叉反应性的方法包括实施例中所述的方法以及本领域已知的标准测定法，例如通过使用生物光干涉，或流式细胞术技术。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(K D)代表，平衡解离常数是解离速率常数和结合速率常数(分别是k dis和k on)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“高亲和力”对于IgG抗体指，该抗体以1x 10 -7M或更低、优选地5x 10 -8M或更低、更优选地大约1x 10 -8M或更低、甚至更优选地大约5x 10 -9M或更低的K D，与靶抗原结合。然而，“高亲和力”结合可以随抗体同种型而变。例如对于IgM同种型，“高亲和力”指抗体具有1x 10 -6M或更低、优选地1x 10 -7M或更低、更优选地大约1x 10 -8M或更低的K D。
与结合例如TNFR2的抗原的参考抗体“竞争结合的抗体”是指这样的抗体，所述抗体在竞争检验中阻断参考抗体与抗原(例如TNFR2)结合的50％或更多，并且反过来，参考抗体在竞争检验中阻断该抗体与抗原(例如TNFR2)结合的50％或更多。示例性竞争检验描述于： “Antibodies”,Harlow and Lane(Cold Spring Harbor Press,Cold Spring Harbor,NY)。竞争结合的抗体可以与参考抗体结合相同的表位区，例如相同表位、相邻表位或重叠表位。
抑制(例如竞争性抑制)参照抗体与其抗原的结合的抗体是指这样的抗体，其抑制50％、60％、70％、80％、90％或95％以上的所述参照抗体与其抗原的结合。反言之，参照抗体抑制50％、60％、70％、80％、90％或95％以上的该抗体与其抗原的结合。抗体与其抗原的结合可以亲和力(例如平衡解离常数)衡量。测定亲和力的方法是本领域已知的。
与参照抗体显示相同或相似的结合亲和力和/或特异性的抗体是指这样的抗体，其能够具有参照抗体的至少50％、60％、70％、80％、90％或95％以上的结合亲和力和/或特异性。这可以通过本领域已知的任何测定结合亲和力和/或特异性的方法进行测定。
本文中的术语“Fc区”用于定义含有至少一部分的恒定区的免疫球蛋白重链的C-端区域。该术语包括天然序列Fc-区和变体Fc-区。在一个实施方案中，人IgG重链Fc-区从重链的Cys226或从Pro230延伸至羧基端。然而，Fc-区的C-端赖氨酸(Lys447)可以存在或可以不存在。除非本文中另外指出，Fc-区或恒定区中的氨基酸残基的编号根据EU编号系统，也称为EU索引，如Kabat，E.A.等，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health，Bethesda，MD(1991)，NIH Publication 91-3242中所述。
术语“效应子功能”是指，可归因于抗体的Fc-区的那些生物活性，其随抗体类别而改变。已知存在五种主要的抗体类别：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以进一步分为亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。IgGFc区可以介导几种重要的效应子功能，例如细胞因子诱导、ADCC、吞噬作用、补体依赖性细胞毒性(CDC)、以及抗体和抗原-抗体复合物的半衰期/清除速率。在一些情况下，取决于治疗目的，这些效应子功能对于治疗性抗体是理想的，但在其他情况下可能是不必要的或甚至是有害的。因此，在一个实施方案中，本发明提供在Fc区中具有氨基酸残基改变从而改变了抗体效应子功能的变体Fc区及其筛选方法。例如，可以在抗体的Fc区中替换至少一个氨基酸残基，从而改变抗体的效应子功能。
“ADCC”是指抗体依赖性细胞介导的细胞毒性。ADCC在人体中主要由自然杀伤细胞(NK细胞)介导。在ADCC中，抗体与靶细胞表面上展示的抗原结合，NK细胞表面的FcγRIIIA识别抗体的Fc区，从而NK细胞被激活，释放穿孔素和颗粒溶解酶，导致靶细胞的裂解和凋亡。评价目标分子的ADCC活性的体外测定试验的非限制性实例描述于US5,500,362(也可以参见，例如Hellstrom，I.等，Proc.Nat’l Acad.Sci.USA 83(1986)7059-7063；和Hellstrom，I.等，Proc.Nat’l Acad.Sci.USA 82(1985)1499-1502)；US 5,821,337(也可以参见Bruggemann，M.等，J.Exp.Med.166(1987)1351-1361)。或者，可采用非放射性测定方法(例如，用于流式细胞术的ACTITM非放射性细胞毒性测定(CellTechnology，Inc.Mountain View，CA) 和
Figure PCTCN2022074755-appb-000001
非放射性细胞毒性测定(Promega，Madison，WI))。适用于这些测定的效应细胞包括外周血单核细胞(PBMC)和自然杀伤(NK)细胞。备选地或另外地，可以在体内评价目标分子的ADCC活性，例如，在如Clynes，R.等，Proc.Nat’lAcad.Sci.USA 95(1998)652-656中公开的动物模型中评价。
“CDC”是指补体依赖性细胞毒性。在CDC中，抗体的Fc区与补体分子C1q结合，继而形成膜攻击复合物，导致靶细胞的清除。参见，例如Liszewski和Atkinson,ch.26,Fundamental immunology,第3版,Paul编，Raven Press,New York,1993,pp917-940。
“ADCP”是指抗体依赖性细胞介导的吞噬作用。在Fc受体介导的该过程中，与抗体结合的靶细胞被吞噬细胞例如巨噬细胞、单核细胞、中性粒细胞和树突细胞所吞噬。多种Fc受体可以参与该过程。Richards等,Mol.Cancer Ther.7(8):2517-2527(2008)描述了用于ADCP的体外试验。
与抗体相关的术语“变体”在本文中指，与参考抗体相比，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域的抗体，其中变体基本上保持改变之前的抗体分子的至少一个生物学特性(例如，抗原结合能力)。目标抗体区域可以是抗体全长、或重链可变区或轻链可变区或其组合、或(一个或多个)重链CDR区或(一个或多个)轻链CDR区或其组合。在本文中，相对于参考抗体区域具有氨基酸改变的抗体区域，也称作该抗体区域的“变体”。
在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在本发明中，就抗体序列而言，氨基酸序列同一性百分数通过将候选抗体序列与参考抗体序列最佳比对后，在一个优选方案中按照Kabat编号规则进行最佳比对后，予以确定。在本文中，在不指定比较窗(即待比较的目标抗体区域)的情况下，将适用于在参考抗体序列的全长上进行比对。在一些实施方案中，就抗体而言，序列同一性可以分布在整个重链可变区和/或整个轻链可变区上，或序列百分数同一性可以仅限定于构架区，而对应CDR区的序列保持100％相同。
类似地，就抗体序列而言，基于比对，可以确定相对于参考抗体在目标抗体区域具有氨基酸改变的候选抗体。
在本发明中，“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。可以通过本领域已知的标准方法，例如定点诱变和PCR介导的诱变，将氨基酸修饰如取代引入本发明的抗体中。
提供功能上相似氨基酸的保守性置换表是本领域熟知的。在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示的优选保守取代残基。
表A
原始残基 示例性取代 优选的保守取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
本发明的各方面将在下面各小节中进一步详述。
I.本发明的抗TNFR2抗体
本发明一方面提供特异性地结合TNFR2，优选人TNFR2蛋白质(例如SEQ ID NO:60,NM_001066.2的人TNFR2序列)的抗体或其抗原结合片段，尤其是人源化抗体或其抗原结合片段。在一些实施方案中，本发明抗体的抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体例如scFv、(Fab’) 2片段、单结构域抗体、双抗体(dAb)或线性 抗体。
抗体的有利生物性质
在一些实施方案中，本发明的抗TNFR2抗体或其抗原结合片段以高亲和力与人TNFR2结合，例如，解离平衡常数(K D)小于或等于大约50x 10 -9M，更优选地大约1-30x 10 -9M,更优选地小于或等于大约5x 10 -9M,例如大约2nM,1.5nM,1.0nM,0.5nM,0.3nM,0.2nM。优选地，K D通过使用生物光干涉测定法(例如Fortebio亲和测量法)测定。在一些实施方案中，本发明的抗TNFR2抗体或其抗原结合片段与人TNFR2结合的解离常数，例如通过Fortebio亲和测量法确定，为大约1-20x 10 -4s -1,例如，大约2x 10 -4s -1,大约5x 10 -4s -1,大约10x 10 -4s -1,大约15x 10 -4s -1,大约20x 10 -4s -1。
在一些实施方案中，本发明的抗TNFR2抗体或其抗原结合片段与猴TNFR2交叉反应。在一些实施方案中，抗体以高亲和力与食蟹猴TNFR2结合，其中K D值(例如通过Fortebio亲和测量法)为小于或等于大约50x 10 -9M，更优选地大约1-30x 10 -9M,更优选地小于或等于大约5x 10 -9M。
在一些实施方案中，本发明抗体或其抗原结合片段以高亲和力结合细胞表面表达的TNFR2。在一个实施方案中，所述表面表达人TNFR2的细胞是肿瘤细胞(例如淋巴瘤细胞，例如K562细胞)、Treg细胞(例如，人外周血天然CD4 +CD25 +CD127 LOW调节性T细胞)、或重组表达TNFR2的哺乳动物细胞。优选地，以流式细胞术(例如FACS)测定，抗体与表达人TNFR2的细胞结合的EC50值。在一些实施方案中，本发明抗体的细胞表面TNFR2结合EC50值小于10nM，例如0.1-5nM,例如在重组表达TNFR2的CHO细胞上通过流式细胞术测量。在另一些实施方案中，本发明抗体的细胞表面TNFR2结合EC50值小于100pM，例如20-60pM，例如在K562细胞或人外周血Treg细胞上通过流式细胞术测量。本领域技术人员明了，在一些情况下，细胞表面TNFR2的密度将影响抗体与细胞的结合EC50测量值。
在一些实施方案中，本发明抗体或其抗原结合片段阻断TNFR2与其配体TNFα的结合。可以以流式细胞术(例如FACS)测定抗体阻断人TNFR2(细胞上表达的TNFR2)与人TNFα结合的能力(如IC50值和最大阻断活性)。优选地，本发明抗体能够完全阻断人TNFR2(细胞上表达的TNFR2)与人TNFα结合。在一些实施方案中，采用FACS测量，本发明抗体的IC50值小于大约10nM，例如大约1,2,3,4,5,6nM。
在一些实施方案中，本发明抗体或其抗原结合片段降低由TNFα结合TNFR2引起的NF-κB信号通路激活。在一些实施方案中，通过荧光报告基因试验(如实施例的Jurkat NF-κB荧光报告试验)，检测抗体降低由TNFα结合TNFR2引起的NF-κB信号通路激活的能力(如，最大抑制和IC50值)。在一些实施方案中，抗体阻断由TNFα结合TNFR2引起的NF-κB信号通路激活，与无抗体加入相比，可以达到3倍以上的抑制。在一些实施方案中，抗体的IC50值小于大约0.1-10nM。
在一些实施方案中，本发明抗体或其抗原结合片段与细胞TNFR2结合在NK和/或效应T细胞存在下激活ADCC信号通路。优选，本发明抗体能够通过ADCC活性杀伤TNFR2阳性肿瘤细胞。在一些实施方案中，通过荧光报告基因试验(如实施例的Jurkat ADCC萤光素酶报告基因检测试验)，检测抗体结合TNFR2引起的ADCC荧光信号的能力(如，最大激活作用和EC50值)。
在一些实施方案中，本发明的抗体或其抗原结合片段抑制表达人TNFR2的肿瘤的生长。在一个实施方案中，肿瘤细胞是表达人TNFR2的淋巴瘤细胞，例如慢性髓系白血病细胞，或是表达人TNFR2的实体瘤细胞，例如结肠癌细胞。在再一实施方案中，本发明抗体或其抗原结合片段引起ADCC依赖性肿瘤细胞杀伤活性。在另一些实施方案中，本发明抗体或其抗原结合片段抑制(例如肿瘤微环境中)表达TNFR2的免疫抑制性调节Treg细胞的功能。
在一些实施方案中，本发明抗体或其抗原结合片段具有如下至少一种或优选地两种作用：
-作用于免疫抑制性Treg细胞，抑制其增殖，从而有利于宿主的免疫功能恢复；
-直接作用于表面表达TNFR2的癌症细胞，并引起ADCC依赖性肿瘤细胞杀伤活性。
在一些实施方案中，本发明抗体或其抗原结合片段在受体者(例如荷瘤动物模型，如实施例的荷瘤小鼠)中达到至少10％肿瘤抑制率，例如至少20％-50％或更高的肿瘤抑制率。
优选地，本发明抗体或其抗原结合片段表现出上述性质中的至少一个、更优选地至少二个、更优选地至少三个、四个、或五个，甚至更优选地上述所有性质。
抗体CDR区
“互补决定区”或“CDR区”或“CDR”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。
下表B中给出了本发明的一些示例性抗体的VH和VL序列组合：
Figure PCTCN2022074755-appb-000002
本领域公知多种用于在一个给定的VH或VL氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196:901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。根据不同的CDR确定方案，这些HVR中的每一个HVR/CDR的残基如下所述。
Figure PCTCN2022074755-appb-000003
Figure PCTCN2022074755-appb-000004
HVR也可以是根据Kabat编号系统位于如下Kabat残基位置的HVR序列：
VL中的位置24-36或24-34(LCDR1)，位置46-56或50-56(LCDR2)，和位置89-97或89-96位置(LCDR3)；和VH中的位置26-35或27-35B(HCDR1)，位置50-65或49-65(HCDR2)，和位置93-102、94-102或95-102(HCDR3)。
HVR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”或“HVR”或“HVR序列”涵盖以上述任一种方式确定的HVR或CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一个优选的实施方案中，本发明CDR序列如图15所示。
下表C给出了本发明的一些示例性CDR序列组合：
Figure PCTCN2022074755-appb-000005
Figure PCTCN2022074755-appb-000006
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
在一些实施方案中，本发明的抗体有至少一个、两个、三个、四个、五个或六个CDR与表B所列任一抗体的可变区序列中的对应CDR相同，或是其变体。在一些实施方案中，本发明的抗体有至少一个、两个、或三个HCDR与表B所列任一抗体的可变区序列中的对应重链CDR相同，或是其变体。在一些实施方案中，本发明的抗体有至少一个、两个、或三个LCDR与表B所列任一抗体的可变区序列中的对应轻链CDR相同，或是其变体。在本文中，“对应CDR”是指，在最佳比对后，在候选抗体的可变区氨基酸序列中与参考抗体的CDR位于最相似位置上的CDR。在本文中，CDR变体是已经通过至少一个，例如1或2或3个氨基酸取代、缺失和/或插入而修饰的CDR，其中包含CDR变体的抗原结合分子基本上保持包含未修饰CDR的抗原结合分子的生物学特性，例如，保持至少60％,70％,80％,90％,或100％的生物学活性(例如抗原结合能力)。可以理解，各CDR可以单独修饰或组合修饰。优选地，氨基酸修饰为氨基酸取代，尤其是保守氨基酸取代，例如表A中列出的优选保守氨基酸置换。
此外，本领域已知，CDR3区，独立于CDR1和/或CDR2区，单独可以确定抗体对关联抗原的结合特异性。并且，可以基于共同CDR3序列，产生具有相同结合特异性的多种其它抗体。
因此，在一个实施方案中，本发明抗体包含来自表B所示任一抗体的重链和/或轻链可变区序列的CDR3序列，其中所述抗体能够特异地结合人TNFR2。在再一实施方案中，所述抗体还可以包含来自同一抗体的重链和/或轻链可变区的CDR2，或来自不同的TNFR2抗体的重链和/或轻链可变区的CDR2。在再一实施方案中，所述抗体还可以包含来自同一抗体的重链和/或轻链可变区的CDR1，或来自不同的TNFR2抗体的重链和/或轻链可变区的CDR1。可以通过本文描述的测定方法，表征这些抗体的活性，包括与人TNFR2的结合活性、阻断TNFR2与TNFα分子结合的活性、和/或抑制肿瘤生长的活性。
再一方面，考虑到抗原结合特异性主要由CDR1、2和3区提供，在一些实施方案中，可以将VH CDR1、2和3序列和VL CDR1、2和3序列“混合并匹配”(即，可以混合并匹配来自结合同一TNFR2抗原的不同抗体的CDR，不过每种抗体优选地含有VH CDR1、2和3和VL CDR1、2和3)，以产生结合TNFR2的本发明其他分子。可以使用本领域已知的结合测定法(例如，ELISA、SET、Biacore)和实施例中描述的那些测定法，测试这类“混合和匹配的”抗体与TNFR2的结合。当混合并匹配VH CDR序列时，来自特定VH序列的CDR1、CDR2和/或CDR3序列优选地替换为结构上相似的CDR序列。同样，当混合并匹配VL CDR序列时，来自特定VL序列的CDR1、CDR2和/或CDR3序列优选地替换为结构上相似的CDR序列。可 以在本发明表B所示抗体之间进行CDR的“混合和匹配”。此外，本领域技术人员明了，也可以通过用来自其它不同抗体的一个或多个VH和/或VL CDR区序列，置换本文中所示抗体的结构上相似的CDR序列，以产生本发明的其它抗体。
因此，在一些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区，所述重链可变区包含重链互补决定区3(HCDR3)，所述HCDR3：
(i)与表B列出的任一抗体的重链可变区的HCDR3相同；或
(ii)与表C所列任一HCDR3序列相同；或
(iii)相对于(i)或(ii)的HCDR3，包含至少1个(优选1-2个或更优选1个)氨基酸改变(优选取代、更优选保守取代)。
在一些实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，且所述抗体的重链互补决定区3(HCDR3)和轻链互补决定区3(LCDR3)：
(i)与表B列出的任一抗体的重链和轻链可变区序列的HCDR3和LCDR3相同；或
(ii)与表C所列任一组合中的HCDR3和LCDR3序列相同；或
(iii)相对于(i)或(ii)的HCDR3和LCDR3，共包含至少1个(优选1-2个或更优选1个)氨基酸改变(优选取代、更优选保守取代)。
在一个实施方案中，本发明抗体或其抗原结合片段包含重链可变区(VH)，其中所述VH包含：
(i)表B所列任一抗体的VH序列中所含的HCDR1、HCDR2和HCDR3序列；或
(ii)表C所列任一组合中的HCDR1、HCDR2和HCDR3序列；或
(iii)相对于(i)或(ii)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2、或1个氨基酸改变(优选氨基酸取代，优选保守取代)的序列。
在另一个实施方案中，本发明抗体或其抗原结合片段包含轻链可变区(VL)，其中所述VL包含：
(i)表B所列任一抗体的VL序列中所含的LCDR1、LCDR2和LCDR3序列；或
(ii)表C所列任一组合中的LCDR1、LCDR2和LCDR3序列；或
(iii)相对于(i)或(ii)的序列，在所述三个CDR区上共包含至少一个且不超过5、4、3、2、或1个氨基酸改变(优选氨基酸取代，优选保守取代)的序列。
在另一个实施方案中，本发明抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述抗体包含：
(i)与表B所列任一抗体的VH和VL序列中所含的6个CDR序列；或
(ii)表C所列任一组合中的6个CDR序列；或
(iii)相对于(i)或(ii)的序列，在6个CDR区上共包含至少一个且不超过10或5、4、3、2、1个氨基酸改变(优选氨基酸取代，优选保守取代)的序列。
在一个实施方案中，本发明抗体或其抗原结合片段包含：
(i)如SEQ ID NO:19所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:20所示的轻链可变区的LCDR1、2和3序列，或者
(ii)如SEQ ID NO:21所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:22所示的轻链可变区的LCDR1、2和3序列，或者
(iii)如SEQ ID NO:23所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:24 所示的轻链可变区的LCDR1、2和3序列，或者
(iv)如SEQ ID NO:25所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:26所示的轻链可变区的LCDR1、2和3序列，或者
(v)如SEQ ID NO:27所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:28所示的轻链可变区的LCDR1、2和3序列，或者
(vi)如SEQ ID NO:29所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:30或31所示的轻链可变区的LCDR1、2和3序列，或者
(vii)如SEQ ID NO:64所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:65所示的轻链可变区的LCDR1、2和3序列。
在一个优选实施方案中，本发明抗体或其抗原结合片段包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中
(i)HCDR1包含SEQ ID NO:1的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:2的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:3的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:4的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:5的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:6的氨基酸序列或由其组成；或者
(ii)HCDR1包含SEQ ID NO:7的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:8的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:9的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:10的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:11的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:12的氨基酸序列或由其组成；或者
(iii)HCDR1包含SEQ ID NO:13的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:14的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:15的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:16的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:17的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:18的氨基酸序列或由其组成。
在一个优选实施方案中，本发明抗体或其抗原结合片段包含表C所列组合之一的6个CDR序列。
抗体可变区
“可变区”或“可变结构域”是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。重链可变区(VH)和轻链可变区(VL)可以进一步再划分为高变区(HVR，又称作互补决定区(CDR))，其间插有较保守的区域(即，构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。在一些情况下，单个VH或VL结构域足以赋予抗原-结合特异性。此外，结合特定抗原的抗体可以使用来自结合所述抗原的抗体的VH或VL结构域筛选互补VL或VH结构域文库而分离(参见，例如，Portolano，S.等，J.Immunol.150(1993)880-887；Clackson，T.等，Nature 352(1991)624-628)。
本领域已知，可以对两个可变区之一或两者(即VH和/或VL)中的一个或多个残基进行修饰，例如，对一个或多个CDR区和/或对一个或多个构架区进行残基修改，尤其是保守残基取代，而修饰后的抗体仍基本上保持改变之前的抗体分子的至少一个生物学特性(例如，抗原结合能力)。例如，可以突变CDR区的残基，改善抗体的一种或多种结合性质(例如亲和性)。 可以在体外或体内测定试验中，评估突变后的抗体的抗原结合性质或其它功能性质。优选地，引入保守替代。优选地，在CDR区中引入的残基改变不超过1个、2个、3个、4个或5个。此外，可以突变构架区残基，例如以改善抗体的性质。例如，可以将一个或多个构架残基“回复突变”为对应的种系序列残基。
CDR移植是本领域已知的另一种抗体可变区修饰方式。由于CDR序列负责大多数抗体-抗原相互作用，故可以构建模拟已知抗体的性质的重组抗体变体。在该抗体变体中，来自已知抗体的CDR序列被移植到具有不同性质的不同抗体的构架区上。因此，在一个实施方案中，本发明涉及这样的抗TNFR2抗体或其抗原结合片段，所述抗体包含来自表B抗体之一的重链和轻链可变区的CDR序列，但具有不同的构架区序列。可以从公共DNA数据库，包括种系抗体基因序列，或从公开文献报道的TNFR2抗体序列，获得用于替换的构架区序列。例如，可以从GenBank数据库获得编码人重链和轻链可变区基因的种系DNA。可以将抗体蛋白序列与数据库中的蛋白序列，使用序列相似性检索工具，例如Gapped BLAST，进行比较。优选，用于替代的构架序列，与选择进行改变的本发明抗体的构架序列，具有结构相似性，例如，具有序列同一性至少80％，85％，90％，或95％、96％、97％、98％、99％以上的构架序列。在一些实施方案中，可以按照实施例9的方式进行抗体的人源化。
在再一实施方案中，可以“混合并匹配”来自本发明示例性抗体(表B所示抗体之一)与其它不同抗TNFR2抗体(优选地，表B所示另一抗体)的VH和VL序列，以产生结合TNFR2的本发明其他抗体。在混合和匹配这些链时，优选地，将来自具体VH/VL配对的VH序列替换为结构相似的VH序列。同样，来自特定VH/VL配对的VL序列优选地替换为结构上相似的VL序列。可以使用本领域已知的结合测定法(例如，ELISA，和实施例部分中描述的其他测定法)测试这类“混合和匹配的”抗体与TNFR2的结合。
因此，在一个实施方案中，本发明的抗体包含表B所列任一抗体的重链可变区VH序列，或由所述氨基酸序列组成。在再一实施方案中，本发明的抗体包含所述VH序列的变体。
在另一个实施方案中，本发明的抗体包含表B所列任一抗体的轻链可变区VL序列，或由所述氨基酸序列组成。在再一实施方案中，本发明的抗体包含所述VL序列的变体。
在再一实施方案中，本发明的抗体包含：
(i)包含SEQ ID NO:19所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:20所示的氨基酸序列的VL序列或其变体，或者
(ii)包含SEQ ID NO:21所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:22所示的氨基酸序列的VL序列或其变体，或者
(iii)包含SEQ ID NO:23所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:24所示的氨基酸序列的VL序列或其变体，或者
(iv)包含SEQ ID NO:25所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:26所示的氨基酸序列的VL序列或其变体，或者
(v)包含SEQ ID NO:27所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:28所示的氨基酸序列的VL序列或其变体；
(vi)包含SEQ ID NO:29所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:30或21所示的氨基酸序列的VL序列或其变体；
(vii)包含SEQ ID NO:64所示的氨基酸序列的VH序列或其变体，和/或包含SEQ ID NO:65所示的氨基酸序列的VL序列或其变体。
在一个实施方案中，VH序列的变体在氨基酸序列上，与参考VH序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性。在一个实施方案中，VH序列的变体在氨基酸序列上，与参考VH序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，包含至少一个且不超过30个、10个、或5、4、3、2、1、0氨基酸改变(优选氨基酸取代，优选保守取代)。优选地序列差异不发生在CDR区中。
在一个优选的实施方案中，VL序列的变体在氨基酸序列上，与参考VL序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性。在一个优选的实施方案中，VL序列的变体在氨基酸序列上，与参考VL序列相比，(优选地，在全长上或在CDR1、2和3三个区域上)，包含至少一个且不超过30个、10个、或5、4、3、2、1、0氨基酸改变(优选氨基酸取代，优选保守取代)。优选地序列差异不发生在CDR区中。
在一个优选的实施方案中，本发明的抗体包含表B所列任一抗体的重链可变区和轻链可变区VH/VL序列对，或由所述氨基酸序列对组成。本发明也提供该抗体的变体，例如在VH、VL、或VH和VL上具有至少95-99％同一性或包含不超过10个氨基酸改变的变体。
在上述任一实施方案中，优选地，相对于参考抗体，抗体变体的重链可变区在1个或多个CDR(优选全部3个CDR)区域上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
在上述任一实施方案中，优选地，相对于参考抗体，抗体变体的轻链可变区VL在1个或多个CDR(优选全部3个CDR)区域上包含不超过10个，优选不超过5个(例如，3、2、1或0个)氨基酸改变(优选氨基酸取代，优选保守取代)。
抗体重链和轻链
在一个实施方案中，本发明的抗体包含重链恒定区和/或轻链恒定区。在一些实施方案中，本发明抗体包含重链Fc区，例如IgG1,IgG2，IgG3或IgG4同种型的Fc区。在另一些实施方案中，本发明抗体含有IgG1-Fc区，尤其是人IgG1-Fc区。在再一些实施方案中，本发明抗体包含κ轻链恒定区，例如人κ轻链恒定区。
取决抗体的期望应用，抗体可以包含相对于天然Fc区具有相同或改变的效应子功能的重链恒定区。示例性“效应子功能”包括C1q结合、补体依赖性细胞毒性(CDC)、Fc受体结合、FcγR介导的效应子功能例如ADCC和抗体依赖性细胞介导的吞噬作用(ADCP)。一般，当抗体的可变区与细胞(例如肿瘤细胞)表面抗原结合以及抗体的Fc区与效应细胞(例如T效应细胞)上的Fc受体结合时，将引起效应子功能。在哺乳动物中，体液免疫大多数通过抗体Fc区与C1q的相互作用以及补体级联来介导。而细胞免疫反应大多数由抗体Fc区与Fcγ受体(即，FcγR)的相互作用来介导。FcγRI,FcγRIIA,FcγRIIIA和FcγRIIIB为激活性FcγR。而FcγRIIB 为抑制性FcγR。激活性受体的细胞内信号传导由受体的胞内ITAM基序的磷酸化介导，这导致效应子功能如ADCC,ADCP、以及通过诱导细胞因子释放引起的炎症反应。抑制性受体FcγRIIB的细胞信号传导通过受体胞内的ITIM基序的磷酸化介导，起到平衡激活性信号传导途径的作用。抗体与FcγR和C1q的相互作用主要取决于铰链和CH2氨基酸序列以及CH2区的糖基化。
在一些实施方案中，因此，本发明抗体包含与激活性FcγR结合的重链恒定区。在一个具体实施方案中，抗体包含结合选自以下的FcR的重链恒定区：FcγRIIIA(CD16a),FcγRIIIA(CD16a)F158变体,FcγRIIIA(CD16a)V158变体。在另一具体实施方案方案中，抗体包含人IgG1或IgG3亚类的恒定区。优选地，本发明抗TNFR2抗体在效应细胞例如NK细胞存在时诱导ADCC。
在一些优选的实施方案中，本发明抗体包含SEQ ID NO:62的人IgG1恒定区氨基酸序列，或相对于SEQ ID NO:62氨基酸序列包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与SEQ ID NO:62的氨基酸序列具有至少95-99％同一性的序列。
在一个优选的实施方案中，本发明抗体包含轻链恒定区。在一个优选实施方案中，轻链恒定区为人κ轻链恒定区。在再一优选实施方案中，轻链恒定区包含SEQ ID NO:63的氨基酸序列，或相对于SEQ ID NO:63的氨基酸序列包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与SEQ ID NO:63的氨基酸序列具有至少95-99％同一性的序列。
在一些优选的实施方案中，本发明抗体包含重链，并且所述重链包含选自SEQ ID NO:32-37和66的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区中，更优选地，不发生在可变区中。
在一些优选的实施方案中，本发明抗体包含轻链，并且所述轻链包含选自SEQ ID NO:38-44和67的氨基酸序列、或相对于其包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或与其具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区中，更优选地，不发生在可变区中。
在一个优选实施方案中，本发明的抗体包含选自以下的重链序列和/或轻链序列：
(a)包含选自SEQ ID NO:32的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:38的氨基酸序列的轻链序列或其变体；
(b)包含选自SEQ ID NO:33的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:39或139的氨基酸序列的轻链序列或其变体；
(c)包含选自SEQ ID NO:34的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:40的氨基酸序列的轻链序列或其变体；
(d)包含选自SEQ ID NO:35的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:41的氨基酸序列的轻链序列或其变体；
(e)包含选自SEQ ID NO:36的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:42的氨基酸序列的轻链序列或其变体；
(f)包含选自SEQ ID NO:37的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:43或44的氨基酸序列的轻链序列或其变体，
(g)包含选自SEQ ID NO:66的氨基酸序列的重链序列或其变体，和/或包含SEQ ID NO:67或44的氨基酸序列的轻链序列或其变体，
其中所述变体与对应的参考序列相比包含至少一个，两个或三个，但不超过20个，10个或5个氨基酸改变的氨基酸序列，或具有至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一性的氨基酸序列。优选地，氨基酸改变不发生在CDR区，更优选地不发生在可变区。
在一些实施方案中，本发明抗TNFR2抗体或其片段的重链和/或轻链还包含信号肽序列。
示例性抗体序列
本发明提供如实施例中分离并表征的特异性结合TNFR2(例如人TNFR2)的抗体，例如鼠源抗体、嵌合抗体和人源化抗体。图16中列出了本发明这些示例性抗体的抗体可变区VH和VL序列。图15中列出了抗体的示例性CDR序列。
抗体变体
在一方面，本发明提供在本文中所述及的任何抗体，尤其是表B所列示例性抗体的变体。在一个实施方案中，抗体变体保持改变前抗体的至少60％,70％,80％,90％,或100％的生物学活性(例如抗原结合能力)。在一些实施方案中，所述改变不导致抗体变体丧失对抗原的结合，但任选地可以赋予诸如提高的抗原亲和力和不同的效应子功能等性质。
可以理解的，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在一些实施方案中，抗体变体与参考抗体在目标抗体序列区域上具有至少80％、85％、90％或95％或99％或更高的氨基酸同一性。例如，在一个实施方案中，本发明抗体，与参考抗体(例如表B所列抗体之一)相比，在3个重链CDR区域上具有至少90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的同一性。在一个实施方案中，本发明抗体，与参考抗体(例如表B所列抗体之一相比)，在3个轻链CDR区域上具有至少90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的同一性。在另一实施方案中，本发明抗体，与参考抗体(例如表B所列抗体之一相比)，在6个CDR区域上具有至少90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的同一性。在再一实施方案中，本发明抗体，与参考抗体(例如表B所列抗体之一)相比，在重链可变区上具有至少80％、85％、90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的序列同一性。在再一实施方案中，本发明抗体，与参考抗体(例如表B所列抗体之一)相比，在轻链可变区上具有至少80％、85％、90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的序列同一性。在再一实施方案中，本发明抗体，与参考抗体(例如表B所列抗体之一)相比，在重链可变区和轻链可变区上具有至少80％、 85％、90％,91％,92％,93％,94％,95％,96％,97％,98％,或99％或更高的序列同一性。
此外，可以对抗体的Fc区进行改变。Fc区的改变可以单独进行，或与上述对构架和/或CDR区的改变相组合。可以改变Fc区，例如，以改变抗体的一种或多种功能，例如血清半衰期，补体固定、Fc受体结合、和/或抗原依赖性细胞毒性。此外，还可以对本发明抗体进行化学修饰(例如，与PEG连接)或改变其糖基化模式。
在另一些实施方案中，取决于期望的抗体应用目的，抗体可以包含经修饰的Fc区变体，所述修饰的Fc区变体相比于未修饰的亲本Fc区具有改变的效应子功能。通常，可以通过将本文所述的可变区连接到含有一个或多个修饰(例如，氨基酸替代、缺失和/或插入)的Fc区上，从而获得具有一种或多种改变(例如增强或降低或消除)的效应子功能的本发明抗TNFR2抗体，例如具有选自如下的增加功能的抗TNFR2抗体：相对于未修饰的亲本Fc区，增加的ADCC、CDC、ADCP、和/或血清半衰期。此外，可以对抗体进行化学修饰，例如改变抗体的糖基化模式，以改变其功能性质。
在某些实施方案中，抗体Fc区可以包含具有一个或多个提高ADCC活性的氨基酸置换的Fc-区，例如，Fc-区的位置298、333和/或334的置换(残基的EU编号)。在一些实施方案中，也可以对Fc-区进行改变，以导致改变的(即，提高的或降低的)C1q结合和/或补体依赖性细胞毒性(CDC)(参见，例如，US6,194,551、WO99/51642和Idusogie，E.E.等，J.Immunol.164(2000)4178-4184)。
在一些实施方案中，抗体包含Fc区变体，所述变体相比于天然未修饰的亲本Fc区，具有增加的ADCC活性。这样的Fc区变体的一些实例包含在选自以下位置的一个或多个氨基酸修饰：234,235,236,238,239,240,241,243,244,245,247,248,249,252,254,255,256,258,262,263,264,265,267,268,269,270,272,276,278,280,283,285,286,289,290,292,293,294,295,296,298,299,301,303,305,307,309,312,313,315,320,322,324,325,326,327,329,330,331,332,333,334,335,337,338,340,360,373,376,378,382,388,389,398,414,416,419,430,433,434,435,436,437,438或439(氨基酸残基根据EU索引编号)。示例性替代包括：236A,239D,239E,268D,267E,268E,268F,324T,332D,和332E。示例性组合替代包括239D/332E,236A/332E,236A/239D/332E,268F/324T,267E/268F,267E/324T,和267E/268F/324T。
已经绘制了人IgG1上与FcγR1，FcγRII，FcγRIII和FcRn的结合位点，并且已经描述了具有改善的结合的变体(参见Shields，R.L。等人(2001)J.Biol.Chem.276：6591-6604)。位置256、290、298、333、334和339处的特定突变显示可改善与FcγRIII的结合。以下组合突变体显示可以改善FcγRIII结合和ADCC活性：T256A/S298A，S298A/E333A，S298A/K224A、S298A/E333A/K334A、S239D/I332E和S239D/I332E/A330L突变。
在另一些实施方案中，可以对抗体Fc进行改变以增加或降低其糖基化程度和/或改变其糖基化模式。对Fc的糖基化位点的添加或缺失可通过改变氨基酸序列以便产生或移除一或多个糖基化位点而方便地实现。举例而言，可实施一或多种氨基酸取代以消除一或多个糖基化位点，由此消除该位点处的糖基化。可制备具有改变类型的糖基化的抗体，例如具有减小量的岩藻糖基残基的低或无岩藻糖化抗体或具有增加的等分GlcNac结构的抗体。这类改变的糖基化模式已显示可增加抗体的ADCC能力。本发明也考虑在与Fc区连接的寡糖中具有至少一个半乳糖残基的抗体变体。这些抗体变体可具有提高的CDC功能。
另外地或可替代地，可通过例如在具有改变的糖基化机制的宿主细胞中表达抗体来实现抗体糖基化程度和/或模式的改变。具有改变的糖基化机制的细胞在本领域中已经被描述，并且可以用作其中表达重组抗体从而产生具有改变的糖基化的抗体的宿主细胞。
在另外一些实施方案中，本发明也考虑具有一些但非所有效应子功能的抗体变体，这使其成为某些应用的理想候选物，在所述应用中抗体的体内半衰期是重要的，但某些效应子功能(如补体和ADCC)是不必要或有害的。例如，Fc区可以包含消除或减弱效应子功能的突变，例如具有突变P329G和/或L234A和L235A的人IgG1Fc区，或具有突变P329G和/或S228P和L235E的人IgG4Fc区。
在某些实施方案中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。例如，可以改变抗体铰链区中的半胱氨酸残基数目，以例如利于轻链和重链的装配或增加或降低抗体的稳定性。残基例如美国专利号5,677,425。
在某些实施方案中，本文中所提供的抗体可进一步经修饰为含有非蛋白质部分。适合抗体衍生的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)，以例如增加抗体的(例如血清)半衰期。用于蛋白质PEG化的方法是本领域已知的，可以将其应用于本发明的抗体。参见例如EP 0154 316和EP 0401384。
II.多核苷酸、载体和宿主
本发明提供编码以上任何抗TNFR2抗体或其片段的核酸。还提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。还提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)。在另一个实施方案中，宿主细胞是原核的。
在一方面，本发明提供编码以上任何抗TNFR2抗体或其片段的核酸。所述核酸可以包含编码抗体的轻链可变区和/或重链可变区的氨基酸序列的核酸，或包含编码抗体的轻链和/或重链的氨基酸序列的核酸。示例性的编码抗体重链可变区的核酸序列包含与选自SEQ ID NO:45,47,49,51,53,55和68的核酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸序列，或者包含选自SEQ ID NO:45,47,49,51,53,55和68的核酸序列。示例性的编码抗体轻链可变区的核酸序列包括与选自SEQ ID NO:46，48，50，52，54，56，57，和69的核酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的核酸序列，或者包括选自SEQ ID NO:46，48，50，52，54，56，57，和69的核酸序列。从适宜的表达载体表达时，由这些多核苷酸编码的多肽能够显示TNFR2抗原结合能力。
本发明中还提供多核苷酸，所述多核苷酸编码来自上文所述的结合TNFR2的抗体的重链VH或轻链VL序列的至少一个CDR区和通常全部三个CDR区。一些进一步的实施方案中，多核苷酸编码上文所述的结合TNFR2的抗体的重链和/或轻链的完整或基本上完整可变区序列。
如本领域技术人员明了的，因为密码子简并性，每一个抗体或多肽氨基酸序列可以由多种核酸序列编码。
在一个优选实施方案中，编码抗体的本发明核酸还包含编码重链恒定区的核苷酸序列， 例如SEQ ID NO:62中所示的恒定区序列或与其基本相同的序列。
在一个优选实施方案中，编码抗体的本发明核酸还包含编码轻链恒定区序列的核苷酸序列，例如SEQ ID NO:63中所示的序列或与其基本相同的序列。
可以采用本领域熟知的方法，通过从头固相DNA合成，或通过PCR诱变，编码结合TNFR2的抗体或其抗原结合片段的上述抗体核酸序列。
在一个实施方案中，提供包含本发明核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。
在一个实施方案中，提供包含所述载体的宿主细胞。用于克隆或表达编码抗体的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，抗体可在细菌中产生，特别当不需要糖基化和Fc效应子功能时。对于抗体片段和多肽在细菌中的表达，见，例如，美国专利号5,648,237，5,789,199和5,840,523，还见Charlton，Methods in Molecular Biology，卷248(B.K.C.Lo，编辑，Humana Press，Totowa，NJ，2003)，第245-254页，其描述抗体片段在大肠杆菌中的表达。在表达后，抗体可以从可溶级分中的细菌细胞糊状物分离，并且可以进一步纯化。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。参见Gerngross，Nat.Biotech.22：1409-1414(2004)，和Li等，Nat.Biotech.24：210-215(2006)。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(293HEK或293细胞，如例如Graham等，J.Gen Virol.36：59(1977)中所描述的)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞(Urlaub等，Proc.Natl.Acad.Sci.USA 77：216(1980))；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。关于适合产生抗体的某些哺乳动物宿主细胞系的综述见例如Yazaki和Wu，Methods in Molecular Biology，卷248(B.K.C.Lo，ed.，Humana Press，Totowa，NJ)，第255-268页(2003)。
III.抗体的制备
在一个实施方案中，提供了制备抗TNFR2抗体的方法，其中所述方法包括，在适合抗体表达的条件下，培养包含编码所述抗体的核酸的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述抗体。为了重组产生抗TNFR2抗体，分离编码抗体(例如上文所描述的抗体)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序(例如通过使用能够与编码抗体重链和轻链的基因特异性结合的寡核苷酸探针进行)。
IV.测定法
可以通过本领域中已知的多种测定法对本文中提供的抗TNFR2抗体鉴定，筛选，或表征 其物理/化学特性和/或生物学活性。
可以通过本领域已知的方法，诸如ELISA，Western印迹等，或本文实施例公开的例示性方法，来测定抗体与人TNFR2的结合。例如，可以使用流式细胞术进行测定，其中抗体与表达人TNFR2的细胞系，例如经转染在细胞表面上表达人TNFR2的CHO细胞，进行反应。其它的细胞也适用于流式细胞术，包括表达人TNFR2的肿瘤细胞或原代Treg细胞。备选地，抗体的结合，包括结合动力学(例如K D值)，可以使用重组TNFR2蛋白，在生物光干涉测定法中测定。在一些实施方案中，使用生物光干涉测定法(例如Fortebio亲和测量)，抗体与人以及其他物种来源的TNFR2的结合平衡解离常数。
针对TNFR2的本发明抑制性/拮抗性抗体可以通过结合细胞膜TNFR2，阻断配体TNFα激活TNFR2信号通路。在一些实施方案中，该阻断活性依赖于抗体对TNFR2与其天然配体TNFα结合的阻断，例如实施例抗体3C4和69B1及其人源化形式、或与其具有相同或相似VH和VL序列的抗体。在另一些实施方案中，该阻断活性不依赖于抗体对TNFR2与其天然配体TNFα结合的阻断，例如实施例抗体72G8及其人源化形式、或与其具有相同或相似VH和VL序列的抗体，这些抗体可以直接抑制细胞表面TNFR2的多聚化，从而抑制TNFR2受体激活。可以使用例如Jurkat TNFR2 NF-κB萤光素酶报告检测系统，在过表达TNFR2的Jurkat细胞中，检测本发明抗体对TNFα激活的TNFR2信号通路的抑制作用。在一些实施方案中，按照实施例8描述的方法，检测本发明抗体对TNFα激活的TNFR2信号通路的抑制作用。
可以通过ADCC报告基因生物活性检测，检测本发明抗体对表达TNFR2的靶细胞的ADCC杀伤作用。ADCC报告基因生物活性检测法选择ADCC作用机制通路激活过程中较早的事件作为检测的读出指标：即效应细胞中由NFAT(活化T细胞核因子)通路介导的基因转录的激活。此外，ADCC报告基因检测法使用工程改造的T细胞作为效应细胞(例如Jurkat细胞)，其中所述细胞稳定表达FcγRIIIa受体(例如V158高亲和力突变体)和由NFAT应答元件驱动表达的萤火虫萤光素酶。在所述检测中，抗体在ADCC作用机制中的生物活性通过NFAT通路活化产生的萤光素酶来定量，而效应细胞中的萤光素酶活性通过生物发光读数来定量。在ADCC报告基因生物活性检测，由此，只有同时具备带有正确表面抗原的靶细胞、正确的特异性抗体及表达FcγRIIIa的效应细胞时，才会获得良好的检测应答。在一个优选的实施方案中，本发明抗体的ADCC活性通过实施例14描述的ADCC报告基因检测方法检测。
本发明还提供了用于鉴定具有生物学活性的抗TNFR2抗体的测定法。生物学活性可以包括例如结合TNFR2(例如结合人TNFR2)，阻断TNFR2(例如结合人TNFR2)与TNFα分子结合、抑制由TNFα结合TNFR2介导的信号传导、引发ADCC活性、和/或抑制肿瘤生长。例如，在体内肿瘤抑制模型中(参见例如实施例15和16)，测试抗体抑制肿瘤生长的能力。在本发明也提供在体内和/或在体外具有此类生物学活性的抗体。
可以理解的是，能够使用本发明的免疫缀合物或多特异性抗体替换或补充抗TNFR2抗体来进行任何上述测定。
V.多特异性抗体
在再一方面，本发明提供特异性地结合TNFR2(优选人TNFR2)的多特异性(包括双特 异性)抗体分子。在一个实施方案中，在多特异性抗体中，本发明的抗体(或其抗原结合片段)形成针对TNFR2的第一结合特异性。在再一实施方案中，所述多特异性抗体还针对以下一种的第二特异性、或还包含针对以下两种分子的第二和第三结合特异性。所述的第二、第三结合特异性例如可以针对肿瘤细胞表面上表达的另一抗原。
在一个实施方案中，结合特异性由抗体的“结合位点”或“抗原结合位点”(抗体分子中与抗原实际结合的区域)提供。在一个优选的实施方案中，抗原结合位点由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)组成的VH/VL对构成。因此，在一个实施方案中，“多特异性”抗体是具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点可以与相同抗原的不同表位或与不同抗原的不同表位结合。有关多特异性抗体及其制备，可以参见例如WO 2009/080251、WO 2009/080252、WO 2009/080253和WO 2010/145793中的描述。
VI.免疫缀合物
再一方面，本发明提供通过将本发明抗体缀合于异源分子而产生的免疫缀合物。在一个实施方案中，在免疫缀合物中，本发明的抗体(或其抗原结合片段)与治疗剂或诊断剂缀合。在一些实施方案中，本发明抗体可以以全长抗体或抗体片段的形式与异源分子缀合。例如，以Fab片段、Fab’片段、F(ab)’2片段、单链scFab抗体、单链scFv等片段形式进行缀合。
可以使用接头来共价连接缀合物的不同实体。适宜的接头包括化学接头或肽接头。有利地的是，接头是利于多肽在递送至靶位点后释放的“可裂解接头”。例如，可以使用酸不稳定性接头、肽酶敏感性接头、光不稳定性接头、二甲基接头或含二硫化物的接头(Chari等，Cancer Research 52(1992)127-131；US 5,208,020)。
适用于缀合物中的治疗剂包括但不限于细胞毒素(例如细胞生长抑制剂或细胞杀伤剂)，药物或放射性同位素。适合于形成免疫缀合物的细胞毒性剂(例如化疗剂)的例子是本领域中已知的，参见例如WO05/103081。例如，细胞毒性剂包括但不限于：放射性同位素；生长抑制剂；酶及其片段如核酸水解酶；抗生素；毒素如小分子毒素或细菌、真菌、植物或动物起源的酶促活性毒素，包括其片段和/或变体；和已知的各种抗肿瘤或抗癌剂。
再一方面，本发明的抗体可以与诊断剂或可检测剂缀合。这类缀合物可以作为临床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可以通过将抗体与可检测剂偶联实现这类诊断和检测，所述可检测剂包括但不限于多种酶，如但不限于辣根过氧化物酶；辅基，如但不限于链霉亲和素/生物素和抗生物素蛋白/生物素；荧光物质；发光物质；放射性物质；和用于各种正电子发射成像术中的正电子发射金属和非放射性顺磁金属离子。
VII.药物组合物和药物制剂
本发明还包括包含抗TNFR2抗体或其免疫缀合物或多特异性抗体的组合物(包括药物组合物或药物制剂)和包含编码抗TNFR2抗体或其免疫缀合物或多特异性抗体的多核苷酸的组合物。这些组合物还可以任选地包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
适用于本发明的药用载体可以是无菌液体，如水和油，包括那些具有石油、动物、植物 或合成起源的，如花生油、大豆油、矿物油、芝麻油等。当静脉内施用药物组合物时，水是优选的载体。还可以将盐水溶液和水性右旋糖以及甘油溶液用作液体载体，特别是用于可注射溶液。合适的药用赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米、面粉、白垩、硅胶、硬脂酸钠、甘油单硬脂酸酯、滑石、氯化钠、干燥的脱脂乳、甘油、丙烯、二醇、水、乙醇等。对于赋形剂的使用及其用途，亦参见“Handbook of PharmaceuticalExcipients”,第五版,R.C.Rowe,P.J.Seskey和S.C.Owen,PharmaceuticalPress,London,Chicago。若期望的话，所述组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂。这些组合物可以采用溶液、悬浮液、乳剂、片剂、丸剂、胶囊剂、粉末、持续释放配制剂等的形式。口服配制剂可以包含标准载体，如药用级甘露醇、乳糖、淀粉、硬脂酸镁、糖精。
可以通过将具有所需纯度的本发明的抗TNFR2抗体、免疫缀合物或多特异性抗体，与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编(1980))混合，来制备包含本发明的药物制剂，优选地以冻干制剂或水溶液的形式。
在本发明的药物组合物和药物制剂中，本发明的抗体可以是唯一的活性剂，或可以与其它治疗剂联合。可以与本发明抗体联合的治疗剂包括但不限于对于待治疗的疾病和/或病症具有有益治疗功效的治疗剂。例如，所述活性成分可以是被治疗的特定适应症所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。例如，可以提供抗癌活性的其它药物成分。本发明抗体与活性成分以对于目的用途有效的量合适地组合存在于药物组合物和药物制剂中。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
关于药物制剂的其它组分，还可以参见WO2015/153513中公开的那些。
VIII.组合产品
在再一方面，本发明还提供了组合产品，其包含本发明的抗体或其抗原结合片段，双特异性抗体或免疫缀合物，以及一种或多种其它治疗剂(例如化疗剂、其他抗体、细胞毒性剂、抗肿瘤药等)。本发明的组合产品可用于本发明的治疗方法中。在一些实施方案中，本发明提供组合产品，其中所述其它治疗剂为例如有效刺激免疫反应从而进一步增强、刺激或上调受试者的免疫反应的治疗剂如抗体。
在一些实施方案中，所述组合产品用于预防或治疗肿瘤。在一些实施方案中，肿瘤为癌症，例如结肠癌、慢性髓系白血病。
IX.方法和用途
在一方面，本发明提供应用本发明TNFR2抗体或其抗原结合片段的方法和用途，例如，体内和体外用于：
(1).阻断TNFR-2与其配体TNFα的结合；
(2).在不阻断TNFR-2与其配体TNFα结合的情况下，直接抑制TNFR2多聚化；
(3)以依赖或不依赖阻断TNFα与TNFR2结合的方式，抑制TNFR2受体激活；
(4)抑制由TNFα结合TNFR2介导的NF-κB信号通路激活；
(5).拮抗TNFR2的天然激动剂TNFα的促Treg细胞增殖作用；
(6)依赖于抗体的Fc区引发ADCC活性，杀伤表面表达TNFR2的肿瘤细胞。
在一些实施方案中，本发明的方法和用途涉及在受试者个体的疾病治疗。在另一些实施方案中，本发明的方法和用途涉及在来自例如受试者的样品中检测TNFR2的存在。在再一些实施方案中，本发明也提供本发明TNFR2抗体或其抗原结合片段在制备用于上述用途的产品(例如药物组合物或药物产品或检测产品)中的用途。
治疗应用
本发明提供了利用本发明抗体或其抗原结合片段在个体或受试者中预防或治疗TNFR2相关疾病的方法和用途，所述疾病包括但不限于，肿瘤，例如结肠癌或慢性髓系白血病。在一些实施方案中，本发明的抗体可以作为唯一活性剂，或可以与其它疗法或治疗剂联合施用。所述的其它疗法和治疗剂包括，例如，靶向肿瘤细胞表面的抗原，通过结合和/或阻断这些分子而消灭肿瘤的药物；激活受试者的免疫系统，促使其自发消灭肿瘤的药物。
在本文中，术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，受试者是人。
在本文中，“治疗”包括1)治疗性措施，该措施治愈、减缓、减轻经诊断的病理状况或疾患的症状及/或停止该经诊断的病理状况或疾患的进展；及2)预防性或防范性措施，该措施预防及/或减缓病理状况或疾患的发展。因此，有治疗需要的受试者包括，已罹患疾患的个体、易于罹患疾患的个体，以及欲预防疾患的个体。所述个体将受益于所述的治疗性措施或预防性措施，并相比于未接受所述处理的个体，表现出在疾病、病症、病状、和/或症状的发生、复发或发展上的减轻或改善。在一些实施方案中，本发明涉及疾病或病症的治疗；在另一些实施方案中，本发明涉及疾病或病症的预防。
在根据本发明的一些实施方案中，疾病或病症的“治疗”是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其至少一个临床症状)。在另一些实施方案中，“治疗”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些生理参数。在另一些实施方案中，“治疗”是指，在身体上(例如，稳定可辨别的症状)、生理上(例如，稳定身体参数)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病或病症的治疗和/或预防效果的方法在本领域中通常是已知的。
在根据本发明的再一些实施方案中，疾病或病症的“治疗”是指疾病或病症的“预防”，包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，受试者是预防性方案的候选者。通常，术语“预防”是指，在疾病或病症的病征或症状发生前(特别是在具有疾病风险的受试者中)的药物施用。
本文术语“治疗有效量”、“预防有效量”或“有效量”，是指本发明的抗TNFR2抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善 一种或多种疾病或病征的症状或该疾病或病症的发展的量。治疗有效量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体单独施用某种活性成分时，治疗有效量仅是指该成分的量。当组合施用一种以上活性成分时，治疗有效量是指引起治疗效果的活性成分的综合量，不论是并行给药、依次给药还是同时给药。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
在一些实施方案中，因此，本发明涉及在受试者中抑制肿瘤的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗TNFR2抗体或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。在另一些实施方案中，本发明也涉及在受试者中增强机体的抗肿瘤免疫应答的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗TNFR2抗体或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。在一些实施方案中，将本发明的抗TNFR2抗体或其抗原结合部分施用于携带肿瘤的受试者，杀伤携带TNFR2的肿瘤。在一些实施方案，将本发明的抗TNFR2抗体或其抗原结合部分施用于携带肿瘤的受试者，刺激抗肿瘤免疫应答。
在另一些实施方案中，本发明提供治疗受试者肿瘤，例如癌症的方法，所述方法包括向所述受试者施用有效量的本文所述的任何抗TNFR2抗体或其片段，或包含所述抗体或片段的免疫缀合物、多特异性抗体，或药物组合物。癌症可以处于早期、中期或晚期或是转移性癌。
可用本发明方法治疗的肿瘤包括但不限于例如结肠癌和慢性髓系白血病。本发明的抗体可以通过靶向细胞表面表达TNFR2的肿瘤细胞和/或Treg细胞，抑制肿瘤的生长。
本发明的抗体(以及包含其的药物组合物或免疫缀合物，以及任何另外的治疗剂)可以通过任何合适的方法给药，包括肠胃外给药，肺内给药和鼻内给药，并且，如果局部治疗需要，病灶内给药。肠胃外输注包括肌内、静脉内、动脉内、腹膜内或皮下给药。在一定程度上根据用药是短期或长期性而定，可通过任何适合途径，例如通过注射，例如静脉内或皮下注射用药。本文中涵盖各种用药时程，包括，但不限于，单次给药或在多个时间点多次给药、推注给药及脉冲输注。
为了预防或治疗疾病，本发明的抗体的合适剂量(当单独或与一种或多种其他的治疗剂组合使用时)将取决于待治疗疾病的类型、抗体的类型、疾病的严重性和进程、所述抗体是以预防目的施用还是以治疗目的施用、以前的治疗、患者的临床病史和对所述抗体的应答，和主治医师的判断力。所述抗体以一次治疗或经过一系列治疗合适地施用于患者。
在上述本发明方法中，可以替代本发明抗体或抗原结合部分，施用本发明的组合物、多特异性抗体或免疫缀合物。或者，在这些方法中，除了施用本发明抗体或抗原结合部分，还可以进一步施用本发明的组合物、多特异性抗体或免疫缀合物。
在一些实施方案中，本发明也提供了，本发明抗TNFR2抗体、组合物、免疫缀合物、 多特异性抗体在制备用于前述方法(例如用于治疗)的药物中的用途。
检测应用
本发明也提供检测样品中TNFR2的方法和试剂盒，其中所述方法包括：(a)将所述样品与本发明抗体或其抗原结合片段或免疫缀合物接触；和(b)检测所述抗体或其抗原结合片段或免疫缀合物和TNFR2蛋白之间复合物的形成。在一些实施方案中，样品来自癌症患者，例如皮肤癌患者。所述检测可以是体外的或体内的。
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是血、血清或生物来源的其他液体样品。在某些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶。在某些实施方案中，待检测的TNFR2是人TNFR2。
在一个实施方案中，抗TNFR2抗体被用于选择适合利用抗TNFR2抗体的治疗的受试者，例如其中TNFR2是用于选择所述受试者的生物标记物。在一个实施方案中，可以使用本发明抗体诊断癌症或肿瘤，例如评价(例如，监测)对象中本文所述疾病(例如，过度增生性或癌性疾病)的治疗或进展、其诊断和/或分期。
在某些实施方案中，提供标记的抗TNFR2抗体。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。示例性标记包括但不限于，放射性同位素32P、14C、125I、3H和131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，荧光素酶(luceriferase)，例如，萤火虫荧光素酶和细菌荧光素酶(美国专利号4,737,456)，荧光素，2，3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，以及利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1.杂交瘤细胞的制备
本实验通过人TNFR2蛋白免疫小鼠，再获取小鼠的脾细胞和骨髓瘤细胞融合，获得能够表达阳性抗体的杂交瘤细胞。
杂交瘤融合
实验动物及免疫方法
Figure PCTCN2022074755-appb-000007
细胞融合：
从免疫后的动物取脾脏，制成脾细胞悬液。经过滤和裂解红细胞后，将脾细胞悬于20ml基础培养基中并计数。
Figure PCTCN2022074755-appb-000008
用20ml基础培养基重悬小鼠骨髓瘤细胞SP2/0细胞(ATCC,CRL-1581)并计数。将SP2/0和脾细胞以1:2～1:1的比例混合，1000rpm离心6min。去除上清后将混合的细胞重悬于10ml融合缓冲液(BTXpress,47-0001)中。再加入15ml融合缓冲液，1000rpm离心5min，去除上清。重复上述步骤一遍后，用适量融合缓冲液重悬细胞，调整混合细胞密度至1×10 7个细胞/ml。电融合仪的参数设置如下。每个电融合皿中加入2ml细胞悬液进行电融合。
Figure PCTCN2022074755-appb-000009
电融合后筛选：
细胞于电融合皿中室温静置5min。将细胞转移入离心管中，用筛选培养基(如下表)稀释细胞至1～2×10 4个细胞/ml。96孔板中每孔加入100μl细胞悬液。融合后第7天更换筛选培养基。培养第10天(或更久，根据细胞生长状态)后进行筛选。通过Elisa检测筛选出表达特异性抗hTNFR2抗体的杂交瘤细胞。
Figure PCTCN2022074755-appb-000010
对筛选的杂交瘤细胞表达的抗体进行PCR测序，确定抗体的可变区基因，进行嵌合抗体筛选，人源化，和亲和力成熟，获得不同阶段候选序列。
本发明示例的3个抗体(3C4,69B1,72G8)及其人源化形式的CDR区、轻链可变区和重链可变区、轻链和重链的氨基酸序列，以及对应的核苷酸序列在下表中列出。
表1：本发明涉及的各抗体相关序列的SEQ ID NO:编号
Figure PCTCN2022074755-appb-000011
Figure PCTCN2022074755-appb-000012
注：HC通过将VH序列融合至SEQ ID NO:62的IgG1部分形成；LC通过将VL序列融合至SEQ ID NO:63的Cκ链形成。
实施例2.嵌合抗体的生产和纯化
(1)GS-CHO细胞中的表达和纯化：
根据制造商的说明书，使用GS Xceed TM Gene Expression System试剂盒(Lonza)产生表达抗体的CHO-S细胞系。首先将抗体分子重链和轻链的DNA序列插入到同一个pCHO1.0质粒中，其中重链在轻链的上游。之后采用化学转染法和电转染法将构建的pCHO1.0质粒转入CHO细胞系，转染48小时之后利用ForteBio检测抗体产量以判断转染效率。转染后的细胞经过两轮加压筛选得到高表达抗体的细胞池(pool)。之后扩增细胞池，大量表达抗体，并收集细胞上清用Protein A纯化上清液，使抗体的纯度＞95％。
(2)HEK293细胞中的表达和纯化：
对于HEK293细胞中抗体的瞬时表达，使用载体pcDNA3.1。首先将编码抗体的重链和轻链的cDNA克隆到pcDNA3.1载体中。使用化学转染的方法将带有抗体分子重链和轻链的载体转入HEK293细胞中。采用的化学转染试剂为PEI(购自Polysciences)，按照生产商提供的方案瞬时转染培养的293HEK(Invitrogen)。转染后，弃去培养基,并用新鲜的EXPI293培养基(Gibco)把细胞稀释到4×10 6/ml。在37℃，5％CO2的条件下培养细胞7天，每48小时流加新鲜培养基。7天后，1300rpm离心20min。取上清液，用Protein A纯化上清液，使抗体的纯度＞95％。
实施例3 生物膜薄层干涉技术测定本发明的嵌合抗体与抗原的结合动力学
本发明采用生物光干涉测量(ForteBio)测定法测定本发明上述3个示例嵌合抗体结合人TNFR2(hTNFR2)，食蟹猴TNFR2(cTNFR2)，鼠TNFR2(mTNFR2)，人TNFR1(hTNFR1)的平衡解离常数(K D)。作为比较，测定阳性对照抗体OPI(WO2017083525中人源化抗体SBT-002e,VH序列SEQ ID NO:58，VL序列SEQ ID NO:59)的平衡解离常数(K D)。
ForteBio亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器(ForteBio)上进行ForteBio亲和测量。再将具有加载的抗体的传感器暴露于100nM的hTNFR2,cTNFR2,mTNFR2,hTNFR1抗原中作用5分钟，之后将传感器转移至分析缓冲液解离5分钟用于解离速率测量。使用1:1结合模型进行动力学的分析。
在如以上测定法所述进行的实验中，3C4,69B1,72G8,以及对照抗体OPI亲和力如表2所示。
表2 anti-TNFR2抗体亲和力
Figure PCTCN2022074755-appb-000013
由以上图表结果可见，上述4个抗体均显示极高的亲和力，其中3C4、69B1和72G8具有与对照抗体OPI相当或较高的亲和力。
实施例4 杂交瘤抗体和过表达人TNFR2的CHO-S细胞(CHOS-hTNFR2)或过表达食蟹猴TNFR2的CHO-S细胞(CHOS-cTNFR2)的结合实验
本研究利用流式细胞仪检测了梯度稀释的本发明的杂交瘤抗体(3C4,69B1,72G8)与表面过表达人TNFR2的CHO-S稳定细胞株的结合情况。
将编码人TNFR2(序列SEQ ID NO:60，NM_001066.2)和食蟹猴TNFR2(序列SEQ ID NO:,61，XP_005544817.1)的cDNA分别克隆到pCHO1.0载体(Invitrogen)上，转染到CHO-S细胞(Invitrogen)，产生过表达人TNFR2的CHO-S细胞(CHOS-hTNFR2)以及过表达食蟹猴TNFR2的CHO-S细胞(CHOS-cTNFR2)。
将CHOS-hTNFR2细胞或CHOS-cTNFR2细胞计数，并稀释至2×10 6个细胞/ml，向U型底96孔板中加入100μl/孔。300g离心5min，去除细胞培养基。将样品(分别是杂交瘤抗体3C4,69B1,72G8以及阳性对照抗体OPI)的稀释物(抗体稀释方法为：最高抗体浓度为300nM，两倍系列稀释在PBS中)加入U型板并重悬细胞，100μl/孔，冰上静置30min。400g离心5min去除上清，PBS洗细胞2遍。300g离心5min去除PBS，每孔加入100μl抗鼠IgG的Alexa Fluor-488标记的二抗(Jackson ImmunoResearch；115-545-072)(1:100稀释于PBS中)，在冰上避光孵育30min。400g离心5min去除上清，PBS洗细胞3遍。用200μl 1×PBS重悬细胞，FACS检测。
在以上测定法所述的实验中，检测结果如图1和2所示，抗体3C4,69B1,72G8均结合CHO-S细胞上过表达的人TNFR2分子(参见图1)和食蟹猴的TNFR2(参见图2)。
实施例5 杂交瘤抗体阻断TNFR2结合TNFα实验
通过ELISA检测了杂交瘤抗体3C4,69B1,72G8以及对照抗体OPI阻断人TNFα与hTNFR2结合的能力。
将hTNFR2蛋白使用PBS重悬并溶解至2ug/ml浓度，包被至酶标板过夜。使用5％BSA封闭1h，将生物素抗原Recombinant Biotinylated TNFα蛋白(ACRO)稀释至3u g/ml，20μl/孔。将如上所述制备的抗体(3C4,69B1,72G8)从最高浓度300nM开始进行梯度稀释，共8个或12个稀释梯度，200μl/孔，于PBS冰上孵育30min。将抗原抗体混合液孵育至酶标板90min，PBS洗三次，弃上清，加1:5000稀释的100μl Avidin-HRP(Invitrogen)/孔，常温30min，加PBS洗六次。使用100ul/孔TMB显色液(solarbio)显色1min，并用终止液(solarbio)100ul/孔终止。酶标仪读数OD 450和OD 620。
实验结果表明，72G8对TNFα配体与受体结合无阻断作用；而3C4,69B1,以及对照抗体OPI的阻断效果完全，IC50值上3C4和69B1优于OPI。(参见图3)
实施例6 嵌合抗体和过表达人TNFR2的CHO-S细胞(CHOS-hTNFR2)或过表达食蟹猴TNFR2的CHO-S细胞(CHOS-cTNFR2)的结合实验
本研究利用流式细胞仪检测了梯度稀释的本发明的嵌合抗体与表面过表达人TNFR2的CHO-S稳定细胞株的结合情况。
将编码人TNFR2(序列SEQ ID NO:60)和食蟹猴TNFR2(序列SEQ ID NO:61)的cDNA克隆分别到pCHO1.0载体(Invitrogen)上，转染到CHO-S细胞(Invitrogen)，产生过表达人TNFR2的CHO-S细胞(CHOS-hTNFR2)以及过表达食蟹猴TNFR2的CHO-S细胞(CHOS-cTNFR2)。
将CHOS-hTNFR2细胞或CHOS-cTNFR2细胞计数，并稀释至2×10 6个细胞/ml，向U型底96孔板中加入100μl/孔。300g离心5min，去除细胞培养基。将样品(分别是嵌合抗体3C4,69B1,,72G8以及阳性对照抗体OPI)的稀释物(抗体稀释方法为：最高抗体浓度为300nM，三倍系列稀释在PBS中)加入U型板并重悬细胞，100μl/孔，冰上静置30min。400g离心5min去除上清，PBS洗细胞2遍。300g离心5min去除PBS，每孔加入100μl抗人Fc的PE标记的二抗(SoutherBiotech；2040-09)(1:100稀释于PBS中)，在冰上避光孵育30min。400g离心5min去除上清，PBS洗细胞3遍。用200μl 1×PBS重悬细胞，FACS检测。
在以上测定法所述的实验中，检测结果如图所示，嵌合抗体3C4,69B1和72G8均结合CHO-S细胞上过表达的人TNFR2分子(参见图4)，且抗体3C4,69B1,72G8均结合食蟹猴的TNFR2(参见图5)。嵌合抗体3C4与69B1的结合EC50值与阳性对照抗体OPI相当；但72G8的结合明显较弱。
实施例7 嵌合抗体阻断TNFR2结合TNFα实验
通过ELISA检测了嵌合抗体3C4,69B1,72G8以及对照抗体OPI阻断人TNFα与hTNFR2结合的能力。
将hTNFR2蛋白使用PBS重悬并溶解至2ug/ml浓度，包被至酶标板过夜。使用5％BSA封闭1h，将生物素抗原Recombinant Biotinylated TNFα蛋白(ACRO)稀释至2u g/ml，10μl/孔。将如上所述制备的抗体(3C4,69B1,72G8)从最高浓度300nM开始进行梯度稀释，共8个或12个稀释梯度，100μl/孔，于PBS冰上孵育30min。将抗原抗体混合液孵育至酶标板90min，PBS洗三次，弃上清，加1:5000稀释的100μl Avidin-HRP(Invitrogen)/孔，常温30min，加PBS洗六次。使用100ul/孔TMB显色液(solarbio)显色1min，并用终止液(solarbio)100ul/孔终止。与酶标仪读数OD 450和OD 620。
实验结果表明，3C4,69B1,以及对照抗体OPI的阻断效果完全，72G8无阻断效果。IC50值上3C4,69B1抗体优于Benchmark OPI。(参见图6)
实施例8 嵌合抗体抑制TNFR2介导的NF-κB信号通路激活实验
针对TNFR2的抑制性抗体可以通过结合细胞膜TNFR2阻断配体TNFα激活TNFR2信号通路。本研究通过Jurkat TNFR2 NF-κB luciferase reporter系统，在过表达TNFR2的Jurkat细胞中，验证了本发明抗体对TNFα激活的TNFR2信号通路的抑制作用。
检测步骤：
以10％1640培养基为稀释buffer，以5*10 4cell/well将Jurkat TNFR2 NF-κB reporter细胞加入各孔，使细胞孵育在终浓度50ng TNFα及不同浓度抗体条件下(最高浓度20nM，依次1:2等比稀释)，37℃CO2培养箱中孵育6h，每孔加入100ul luciferase底物液并震荡2min。多功能酶标仪(Molecular Device i3)读值。按照如下公式，计算抗体荧光读值的倍数变化：
倍数变化＝以加入抗体的各孔的荧光读值/对照组(无TNFα处理组)的荧光读值。
实验结果(图7)表明，阻断TNFR2结合TNFα的嵌合抗体3C4,69B1均可以抑制TNFα诱导的TNFR2 NF-κB信号通路激活，且3C4和69B1抗体的抑制活性优于阳性对照抗体OPI。此外，令人惊奇的，72G8抗体展示出一种新的TNFR2功能活性抑制机制，该抗体可以在不阻断TNFR2结合TNFα的情况下，直接抑制TNFR2多聚化，从而抑制TNFα诱导的TNFR2NF-κB信号通路激活。
实施例9 嵌合抗体人源化
将得到的抗体3C4和69B1嵌合抗体进行人源化。步骤如下：
①确定CDR环结构；
②在人种系序列数据库为重链和轻链的每个V/J区域找到最接近的同源序列；
③筛选与重链轻链最匹配的人种系以及最低量的回复突变；
④将嵌合抗体的CDR区构建至人的骨架区上；
⑤使用序列和结构特征，确定骨架区中起到维持CDR功能的氨基酸位置；
⑥在确定为重要的序列位置进行回复突变(返回到输入氨基酸类型)；
⑦优化风险位点的氨基酸。
获得的人源化抗体hz3C4.7,hz69B1.5,hz69B1.6,hz69B1.11,hz69B1.20的CDR、轻链可变区和重链可变区，轻链和重链的氨基酸序列请参见所附序列表。
实施例10 人源化抗体和表达人TNFR2的细胞的结合实验
本研究利用流式细胞仪检测了梯度稀释的抗体与表面过表达人TNFR2的CHO-S稳定细胞株的结合情况。
将编码人TNFR2的cDNA克隆到pCHO1.0载体(Invitrogen)上，转染到CHO-S细胞(Invitrogen)，产生过表达人TNFR2的CHO-S细胞(CHOS-hTNFR2)。
将CHOS-hTNFR2细胞计数，并稀释至2×10 6个细胞/ml，向U型底96孔板中加入100μl/孔。300g离心5min，去除细胞培养基。将样品(分别是人源化抗体hz3C4.7,hz69B1.5,hz69B1.6,hz69B1.11,hz69B1.20,嵌合抗体69B1，以及阳性对照抗体OPI)(抗体稀释方法为：最高抗体浓度为300nM，三倍稀释在PBS中)加入U型板并重悬细胞，100μl/孔，冰上静置30min。400g离心5min去除上清，PBS洗细胞2遍。300g离心5min去除PBS，每孔加入100μl抗人Fc的PE标记的二抗(SoutherBiotech；2040-09)(1:100稀释于PBS中)，在冰上避光孵育30min。400g离心5min去除上清，PBS洗细胞3遍。用200μl 1×PBS重悬细胞，FACS检测。
在以上测定法所述的实验中，抗体和CHOS-hTNFR2细胞的结合情况如图8所示。人源化抗体hz3C4.7,hz69B1.5,hz69B1.6,hz69B1.11,hz69B1.20均结合CHO-S细胞上过表达的人TNFR2分子(参见图8)。
实施例11 人源化抗体阻断TNFR2结合TNFα实验
按照实施例7相似的方式，通过ELISA检测了人源化抗体hz3C4.7,hz69B1.5,hz69B1.6,hz69B1.11,hz69B1.20以及对照抗体OPI阻断人TNFα与hTNFR2结合的能力。
结果显示在图9A和图9B中。如图所示，3C4和69B1的人源化形式表现出完全的阻断效果。
实施例12 人源化抗体抑制TNFR2介导的NF-κB信号通路激活实验
按照实施例8相似的方式，通过Jurkat TNFR2 NF-κB luciferase reporter系统，在过表达TNFR2的Jurkat细胞中，检查了3C4和69B1的人源化形式抗体对TNFα激活的TNFR2信号通路的抑制作用。结果显示在图10A和10B中。
实施例13 人源化抗体和K562,Treg细胞的结合实验
本研究利用流式细胞仪检测了梯度稀释的本发明的人源化抗体与K562细胞(ATCC,慢性髓系白血病细胞)和Treg细胞的结合情况。
Treg制备如下：复苏人的PBMC细胞(ALLCELLS，PB005F)，使用流式分选CD4+CD25+CD127low细胞，分离出Treg细胞。按照CD4+：anti-CD3/CD28 Beads＝1:1，加入Dynabeads Human T-Activator CD3/CD28(Gibco，11131D)，并加入重组10ng/mL IL2，刺激7天。
检测步骤：400g，5min离心，去除细胞培养基，PBS重悬K562或Treg细胞，计数后，调整细胞密度为2×10 6个/ml，向U型底96孔板中加入100μl/孔。加入待测抗体，三倍梯度稀释，冰上静置30min。300g，5min去除上清，PBS洗细胞1遍。300g，5min去除PBS，每孔加入100μl 1:200稀释的PE-抗人Fc抗体(SOUTHERN BIOTECH,2040-09)。冰上避光孵育30min。400g，5min去除上清，PBS洗细胞2遍。用100μl PBS重悬细胞，流式细胞仪(BD，ACCURIC6 plus)检测。
在以上试验中，结果如图11所示，人源化抗体hz3C4.7,hz69B1.5,hz69B1.11,hz69B1.20结合K562和Treg细胞与对照抗体OPI相比，具有更优的结合能力。
实施例14 人源化抗体的ADCC reporter实验
本研究利用Jurkat ADCC reporter实验(Promega，G7102)检测了梯度稀释的本发明的人源化抗体对CHOS-hTNFR2细胞的ADCC杀伤作用。
检测步骤：根据供应商提供的实验方案,梯度稀释抗体，起始浓度为50nM，1:2等比稀释。之后，每孔加入2.5×10 4CHOS-hTNFR2 cells，和1.75×10 5Jurkat效应细胞。37℃，CO2培养箱孵育20h，加入luciferase底物。2min后使用多功能酶标仪读数(MD，I3多功能酶标仪)检测。
在以上试验中，结果如图12所示，人源化抗体hz3C4.7,hz69B1.5,hz69B1.11,hz69B1.20对表达hTNFR2的CHO-S细胞与对照抗体OPI相比，具有更好的ADCC杀伤效果。
实施例15.抗K562肿瘤药效试验
本实验采用K562细胞接种NOD/SCID小鼠测定本发明的抗TNFR2抗体的抗肿瘤作用。
NOD/SCID小鼠：
雌性NOD/SCID小鼠购自北京维通利华实验动物技术有限公司，等级为SPF级，质检单位为北京维通利华实验动物技术有限公司，合格证编号为NO.11400700380384。小鼠在到达后驯化7天，随后开始研究。
细胞：
K562细胞是人慢性髓系白血病细胞，购自ATCC(CAT#:CCL-243)，并严格按照说明书进行常规传代培养用于后续体内实验。离心收集细胞，在Matrigel中重悬细胞并调整细胞密度为5×10 6个/ml。取0.2ml细胞悬液皮下接种至NOD/SCID小鼠右侧腹部区域中，建立K562荷瘤小鼠模型。
给药：
肿瘤细胞接种6天后检测各只小鼠瘤体积，挑选出瘤体积在25mm 3～118mm 3范围内的小鼠按瘤体积随机分组(每组8只小鼠)，每组肿瘤体积平均在65mm 3左右。给药剂量和方式如表3所示。h-IgG(购自EQUITECH-BIO)作为阴性对照。分别在接种后第6、10、13、17天给药，每周给药2次，共给药四次，并监测小鼠瘤体积与体重。在每次给药前测定体重和肿瘤体积，接种后第27天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定： 采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。采用电子天平测定体重。
表3.实验设计
Figure PCTCN2022074755-appb-000014
肿瘤抑制率结果如图13和表4所示：在接种后第27天，嵌合抗体3C4和69B1单药均能显示肿瘤抑制效果。3C4肿瘤抑制率为37％，69B1肿瘤抑制率分别为33％。因此，本发明针对TNFR2分子的抗体对肿瘤有抑制效果。
表4.第27天肿瘤抑制率
Figure PCTCN2022074755-appb-000015
实施例16.抗MC38-TNFR2肿瘤药效试验
本实验采用MC38-TNFR2细胞(表达人TNFR2的小鼠结肠癌细胞)接种C57BL/6小鼠测定本发明的抗TNFR2抗体的抗肿瘤作用。
C57小鼠：
雌性C57BL/6背景的小鼠购自北京维通利华实验动物技术有限公司，等级为SPF级，质检单位为北京维通利华实验动物技术有限公司，合格证编号为NO.1100111911055796。小鼠在到达后驯化7天，随后开始研究。
细胞：
小鼠MC38-TNFR2细胞自主构建，并严格按照说明书进行常规传代培养用于后续体内实验。离心收集细胞，在PBS中重悬细胞并调整细胞密度为2×10 6个/ml。在第0天取0.2ml细胞悬液皮下接种至人C57BL/6小鼠右侧腹部区域中来建立MC38-TNFR2荷瘤小鼠模型。
给药：
肿瘤细胞接种7天后检测各只小鼠瘤体积，按瘤体积随机分组(每组7只小鼠)，每组肿瘤体积平均在63mm 3左右。给药剂量和方式如表5所示，h-IgG(购自EQUITECH-BIO)作为阴性对照，每周给药2次，共给药四次，并监测小鼠瘤体积与体重。接种后第21天计算相对肿瘤抑制率(TGI％)，计算公式如下：TGI％＝100％*(对照组肿瘤体积–治疗组肿瘤体积)/(对照组肿瘤体积–对照组给药前肿瘤体积)。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。采用电子天平测定体重。
表5.实验设计
Figure PCTCN2022074755-appb-000016
Figure PCTCN2022074755-appb-000017
肿瘤抑制率结果如图14和表6所示：在接种后第21天，hz3C4.7和hz69B1.20单药均能显示肿瘤抑制效果。Hz3C4.7单药肿瘤抑制率为28％，hz69B1.20肿瘤抑制率为41％。因此，本发明针对TNFR2分子的抗体对肿瘤有抑制效果。
表5.第21天肿瘤抑制率
Figure PCTCN2022074755-appb-000018

Claims (18)

  1. 结合TNFR2的抗体或其抗原结合片段，其包含
    (i)如SEQ ID NO:19所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:20所示的轻链可变区的LCDR1、2和3序列，或者
    (ii)如SEQ ID NO:21所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:22所示的轻链可变区的LCDR1、2和3序列，或者
    (iii)如SEQ ID NO:23所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:24所示的轻链可变区的LCDR1、2和3序列，或者
    (iv)如SEQ ID NO:25所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:26所示的轻链可变区的LCDR1、2和3序列，或者
    (v)如SEQ ID NO:27所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:28所示的轻链可变区的LCDR1、2和3序列，或者
    (vi)如SEQ ID NO:29所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:30或31所示的轻链可变区的LCDR1、2和3序列，或者
    (vii)如SEQ ID NO:64所示的重链可变区的HCDR1、2和3序列，以及如SEQ ID NO:65所示的轻链可变区的LCDR1、2和3序列。
  2. 结合TNFR2的抗体或其抗原结合片段，其包含重链可变区的3个互补决定区HCDR，以及轻链可变区的3个互补决定区LCDR，其中：
    (i)HCDR1包含SEQ ID NO:1的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:2的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:3的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:4的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:5的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:6的氨基酸序列或由其组成；或者
    (ii)HCDR1包含SEQ ID NO:7的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:8的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:9的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:10的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:11的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:12的氨基酸序列或由其组成；或者
    (iii)HCDR1包含SEQ ID NO:13的氨基酸序列或由其组成，HCDR2包含SEQ ID NO:14的氨基酸序列或由其组成，HCDR3包含SEQ ID NO:15的氨基酸序列或由其组成，LCDR1包含SEQ ID NO:16的氨基酸序列或由其组成，LCDR2包含SEQ ID NO:17的氨基酸序列或由其组成，且LCDR3包含SEQ ID NO:18的氨基酸序列或由其组成，
    或者，其中所述抗体包含(i)-(iii)的CDR序列组合之一的变体，其中所述变体在6个CDR区上共包含至少一个且不超过5、4、3、2或1个氨基酸改变(优选氨基酸取代，优选保守取代)，优选地重链CDR3保持不变。
  3. 权利要求1或2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    所述重链可变区包含：
    (i)SEQ ID NO:19所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (ii)SEQ ID NO:21所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (iii)SEQ ID NO:23所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (iv)SEQ ID NO:25所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (v)SEQ ID NO:27所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (vi)SEQ ID NO:29所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (vii)SEQ ID NO:64所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  4. 权利要求1或2的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    所述轻链可变区包含：
    (i)SEQ ID NO:20所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (ii)SEQ ID NO:22所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (iii)SEQ ID NO:24所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (iv)SEQ ID NO:26所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，或者
    (v)SEQ ID NO:28所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或者
    (vi)SEQ ID NO:30或21所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列；或者
    (vii)SEQ ID NO:65所示的氨基酸序列、或与其具有至少80％、85％、90％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
  5. 权利要求1至4中任一项的抗体或其抗原结合片段，其包含选自以下的重链可变区和轻链可变区
    (i)包含SEQ ID NO:19所示的氨基酸序列的重链可变区，和包含SEQ ID NO:20所示的氨基酸序列的轻链可变区，或者
    (ii)包含SEQ ID NO:21所示的氨基酸序列的重链可变区，和包含SEQ ID NO:22所示的氨基酸序列的轻链可变区，或者
    (iii)包含SEQ ID NO:23所示的氨基酸序列的重链可变区，和包含SEQ ID NO24所示的氨基酸序列的轻链可变区，或者
    (iv)包含SEQ ID NO:25所示的氨基酸序列的重链可变区，和包含SEQ ID NO:26所示的氨基酸序列的轻链可变区，或者
    (v)包含SEQ ID NO:27所示的氨基酸序列的重链可变区，和包含SEQ ID NO:28所示的氨基酸序列的轻链可变区，或者
    (vi)包含SEQ ID NO:29所示的氨基酸序列的重链可变区，和包含SEQ ID NO:30或31所示的氨基酸序列的轻链可变区，或者
    (vii)包含SEQ ID NO:64所示的氨基酸序列的重链可变区，和包含SEQ ID NO:65所示的氨基酸序列的轻链可变区。
  6. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体是IgG1,IgG2,IgG3,或IgG4形式的抗体或其抗原结合片段，优选人IgG1 Fc区，优选地所述抗体在效应细胞例如NK细胞存在时诱导ADCC活性。
  7. 前述权利要求任一项的抗体或其抗原结合片段，其中所述抗体是鼠源抗体、或嵌合抗体、或人源化抗体，或者其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体、scFv、(Fab’)2片段、单结构域抗体、双抗体(dAb)或线性抗体。
  8. 抗TNFR2抗体或其抗原结合片段，其中所述抗体具有以下一个或多个特性：
    (i)抑制(例如，竞争性抑制)权利要求1-7的任一抗体与人TNFR2的结合；
    (ii)与权利要求1-7的任一抗体结合相同或重叠的表位；
    (iii)与权利要求1-7的任一抗体竞争结合人TNFR2。
  9. 一种分离的核酸，其编码前述权利要求任一项的抗TNFR2抗体或其抗原结合片段。
  10. 一种载体，其包含权利要求9的核酸，优选地所述载体是表达载体。
  11. 一种宿主细胞，其包含权利要求9的核酸或权利要求10的载体，优选地，所述宿主细胞是哺乳动物细胞。
  12. 制备抗TNFR2抗体或其抗原结合片段的方法，所述方法包括在适于表达编码前述权利要求1-8中任一项的抗体或其抗原结合片段的核酸的条件下培养包含所述核酸的宿主细胞，任选地分离所述抗体或其抗原结合片段，任选地所述方法还包括从所述宿主细胞回收所述抗 TNFR2抗体或其抗原结合片段。
  13. 免疫缀合物，其包含与治疗剂或诊断剂缀合的前述权利要求1-8中任一项的抗体或其抗原结合片段。
  14. 包含前述权利要求1-8任一项的抗体或其抗原结合片段的多特异性抗体。
  15. 药物组合物，其包含前述权利要求1-8任一项的抗体或其抗原结合片段或权利要求13的免疫缀合物或权利要求14的多特异性抗体，以及任选地药用辅料。
  16. 前述权利要求1-8任一项的抗体或其抗原结合片段的用途，所述用途选自在体内或在体外用于：
    (1)阻断TNFR-2与其配体TNFα的结合；
    (2)在不阻断TNFR-2与其配体TNFα结合的情况下，直接抑制TNFR2多聚化；
    (3)以依赖或不依赖阻断TNFα与TNFR2结合的方式，抑制TNFR2受体激活；
    (4)抑制由TNFα结合TNFR2介导的NF-κB信号通路激活；
    (5)拮抗TNFα的促Treg细胞增殖作用；和/或
    (6)依赖于抗体的Fc区引发ADCC活性，杀伤表面表达TNFR2的肿瘤细胞。
  17. 在受试者中预防或治疗肿瘤的方法，所述方法包括向所述受试者施用有效量的前述权利要求1-8任一项的抗TNFR2抗体或其抗原结合片段、或权利要求13的免疫缀合物、或权利要求14的多特异性抗体、或权利要求15的药物组合物，优选地，所述肿瘤是结肠癌；或慢性髓系白血病。
  18. 检测样品中TNFR2的方法，所述方法包括
    (a)将样品与前述权利要求1-8中任一项的抗体或其抗原结合片段接触；和
    (b)检测所述抗体或其抗原结合片段和TNFR2间的复合物的形成；任选地，抗体是被可检测地标记的。
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