WO2022068891A1 - Pd-1 antibody, and preparation method therefor and application thereof - Google Patents

Pd-1 antibody, and preparation method therefor and application thereof Download PDF

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Publication number
WO2022068891A1
WO2022068891A1 PCT/CN2021/121785 CN2021121785W WO2022068891A1 WO 2022068891 A1 WO2022068891 A1 WO 2022068891A1 CN 2021121785 W CN2021121785 W CN 2021121785W WO 2022068891 A1 WO2022068891 A1 WO 2022068891A1
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seq
binding protein
amino acid
acid sequence
antibody
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PCT/CN2021/121785
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French (fr)
Chinese (zh)
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刘雅容
种孟阳
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苏州沙砾生物科技有限公司
珠海拓域生物科技有限公司
上海沙砾生物科技有限公司
珠海沙砾生物科技有限公司
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Priority to CN202180007011.0A priority Critical patent/CN114761434B/en
Publication of WO2022068891A1 publication Critical patent/WO2022068891A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein that binds to PD-1.
  • Programmed death-1 is a type I membrane protein with 288 amino acids, mainly expressed on the surface of activated T cells.
  • PD-1 has two ligands, namely programmed death ligand-1 (Programmed Death Ligand-1, PD-L1) and PD-L2.
  • the interaction of PD-1 with PD-L1 and PD-L2 can down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive effect.
  • PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, restore the activity of T cells, and play a role in killing tumor cells, thereby inhibiting tumor growth.
  • immunomodulation targeting PD-1/PD-L1 has important implications for tumor suppression.
  • anti-PD-1/PD-L1 pathway blocking antibody drugs still face many clinical challenges, such as low efficacy, drug resistance and side effects, and it is still necessary to continue to develop more effective anti-PD-1 antibodies .
  • the present application provides an antigen-binding protein that binds PD-1, which exhibits one or more desired functional properties, such as high-affinity binding to PD-L1, the ability to inhibit the binding of PD-1 to PD-1, Enhanced T cell activation includes the ability to proliferate, IFN- ⁇ and/or IL-2 secretion, ability to stimulate antibody responses, and/or ability to reverse the suppressive function of immunosuppressive cells such as T regulatory cells.
  • the present application also provides a scFv-huIgG1 Fc-type antibody, which is convenient to be used in combination with cellular drugs in an autocrine form.
  • the application also provides nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells and methods for preparing the isolated antigen binding proteins.
  • Antigen binding proteins that bind PD-1 disclosed herein can be used (alone or in combination with other active agents or therapeutic modalities) to treat, prevent and/or diagnose diseases, such as cancer diseases (eg, solid and soft tissue tumors).
  • the application provides an isolated antigen-binding protein comprising at least one CDR in the variable VH of an antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO: 7 or 9.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein competes with a reference antibody for binding to PD-1, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, and the reference antibody has a
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:5:
  • the amino acid sequence shown in 6, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1;
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence shown; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the VH of the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the HCDR2 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:2.
  • the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:1.
  • the VH of the isolated antigen binding protein comprises FR1, FR2 and FR3, FR4, wherein the FR1 comprises the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:19.
  • the FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:20.
  • the FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:13 or SEQ ID NO:21.
  • the FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:14 or SEQ ID NO:22.
  • the isolated antigen binding protein comprises VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 7 or 9.
  • the isolated antigen binding protein comprises an antibody heavy chain constant region.
  • the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG constant region.
  • the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG4 constant region and/or a human IgGl constant region.
  • the isolated antigen binding protein comprises at least one CDR in the variable region VL of an antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10.
  • the VL of the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the LCDR2 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:5.
  • the LCDR3 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:6.
  • the VL of the isolated antigen binding protein comprises FR1, FR2 and FR3, FR4, wherein the FR1 comprises the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:23.
  • the FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:16 or SEQ ID NO:24.
  • the FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:25.
  • the FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:18 or SEQ ID NO:26.
  • the isolated antigen binding protein comprises VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 8 or 10.
  • the isolated antigen binding protein has one or more of the following properties:
  • c) can block the binding of PD-1 and PD-L2;
  • d) can stimulate the secretion of IL-2, TNF- ⁇ and/or IFN- ⁇ in immune cells;
  • f) can improve the killing ability of immune cells.
  • the isolated antigen binding protein has one or more of the following properties:
  • c) can block the binding of PD-1 and PD-L2;
  • d) can stimulate the secretion of IL-2 and/or IFN- ⁇ in immune cells
  • e is capable of inhibiting tumor growth and/or tumor cell proliferation.
  • the application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins.
  • the application provides a vector comprising the nucleic acid molecule.
  • the application provides a cell comprising the nucleic acid molecule or the vector.
  • the present application provides a method of preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable adjuvant.
  • the present application provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in preparing a medicine, the Drugs are used to treat diseases or conditions mediated by PD-1.
  • the PD-1 mediated disease or disorder comprises cancer.
  • the present application provides a method for inhibiting the binding of PD-1 to PD-L1, comprising administering an effective amount of the antigen-binding protein, the nucleic acid molecule, and the carrier to a subject in need thereof , the cell and/or the pharmaceutical composition.
  • the present application provides a method for inhibiting the binding of PD-1 to PD-L2, comprising administering an effective amount of the antigen-binding protein, the nucleic acid molecule, and the carrier to a subject in need thereof , the cell and/or the pharmaceutical composition.
  • the present application provides a method of preventing, alleviating or treating a PD-1-mediated disease or disorder, comprising administering to a subject in need thereof an effective amount of the antigen-binding protein, the nucleic acid molecule , the carrier, the cell and/or the pharmaceutical composition.
  • the PD-1 mediated disease or disorder comprises cancer.
  • Figure 1 shows the affinity (based on BLI method) detection curve of the tetravalent human PD-L1 extracellular segment mutant described in the present application and PD-1-huIgG1 Fc;
  • Figure 2 shows the inhibition curves of 6H6 and pembrolizumab huIgG1 antibodies to PD-1 and PD-L1 binding;
  • Figure 3 shows the binding curves of huIgG1 antibodies of 6H6 and pembrolizumab to PD-1.
  • FIGS 4A-4B show the results of the proportion of CD107a cells added to the PD-1 antibody in the tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B.
  • TIL tumor-infiltrating lymphocyte
  • FIGS 5A-5F are graphs showing the results of cytokine secretion by the addition of PD-1 antibody to tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B.
  • TIL tumor-infiltrating lymphocyte
  • Figures 6A-6B show the results of the mixed lymphocyte reaction (MLR) assay to detect the stimulation of T lymphocytes by PD-1 antibody.
  • MLR mixed lymphocyte reaction
  • Figure 7 shows the results of the enhanced killing ability of TCR-T cells by adding PD-1 antibody.
  • PD-1 generally refers to programmed cell death 1, also known as “programmed cell death 1"
  • PD-1 is normally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs) and is involved in apoptosis.
  • PD-1 usually contains an extracellular IgV domain, a transmembrane domain and an intracellular domain.
  • PD-1 binds two ligands, PD-L1 and PD-L2.
  • the "PD-1” includes any native PD-1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rat).
  • PD-1 can exist as a transmembrane protein or as a soluble protein.
  • PD-1 includes complete PD-1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-1, and functional variants, isoforms, derivatives, and analogs of PD-1, Epitope analogs.
  • the amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org), accession number Q15116.
  • the term "PD-L1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which is associated with PD-1 binding downregulates T cell activation and cytokine secretion.
  • P-L1 includes any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rats) ).
  • the term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 produced by cellular processing.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • "PD-L1" includes complete PD-L1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-L1, and functional variants, isoforms, derivatives, and analogs of PD-L1, as well as those having at least one in common with PD-L1 Epitope analogs.
  • the basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • the complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.
  • isolated or purified generally refers to a molecule (eg, antibody, nucleic acid, etc.) that is at least partially separated from other molecules to which it is ordinarily associated in its natural state.
  • An “isolated or purified polypeptide” is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, cellular debris, and growth media.
  • An “isolated or purified nucleic acid” is at least partially separated from the nucleic acid that normally flanks the polynucleotide in its natural state.
  • polynucleotides fused to regulatory or coding sequences to which they are not normally associated are considered isolated in this application, eg, due to recombinant techniques.
  • Such molecules are considered isolated even when present, for example, in the chromosome of a host cell or in a nucleic acid solution.
  • isolated and purified are not intended to refer to the complete absence of such substances or the absence of water, buffers or salts, unless they are present in quantities that interfere with the experimental or therapeutic use of the molecule.
  • the antigen binding proteins of the present application and nucleic acids encoding the antigen binding proteins of the present application are isolated/purified.
  • antigen binding protein is used in its broadest sense and is meant to comprise a moiety that binds to an antigen or target and optionally comprises a framework that allows the antigen binding moiety to adopt a configuration that facilitates binding of the antigen binding protein to the antigen or framework part of the protein.
  • antigen binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; trifunctional antibodies; tetrabodies; Fab fragments; F(ab') 2 fragments; ; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof.
  • Antigen binding proteins can include, for example, alternative protein frameworks or artificial frameworks with grafted CDRs or CDR derivatives.
  • Such frameworks include, but are not limited to: antibody-derived frameworks comprising mutations introduced to stabilize, for example, the three-dimensional structure of the antigen binding protein; and fully synthetic frameworks comprising, for example, biocompatible polymers. See, eg, Korndorfer et al, 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al, Biotechnol. Prog. 20: 639-654 (2004).
  • PAMs peptide antibody mimetics
  • frameworks based on antibody mimetics that utilize fibronectin components as the framework.
  • antibody is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelized single domain antibodies (eg, heavy chain variable domain antibodies).
  • Antibodies generally have the structure of immunoglobulins, and may comprise proteins of at least two heavy (HC) and two light (LC) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • immunoglobulins can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ , ⁇ , ⁇ chains, respectively , ⁇ chain, ⁇ chain.
  • immunoglobulins can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • each light chain comprises a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region contains one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • variable domains of native heavy and light chains each comprise four FR regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), mostly in a ⁇ -sheet configuration, connected by three CDRs, Loop connections are formed, and in some cases part of a beta-sheet structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions and together with the CDRs from the other chain form the antigen-binding site of the antibody.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • variable generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, known as complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portion of the variable domain is called the framework (FR).
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • the more highly conserved portion of the variable domain is called the framework (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, mostly in a ⁇ -sheet configuration, connected by three CDRs, forming loops connecting, and in some cases forming part of, a ⁇ -sheet structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody, the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions , eg involved in antibody-dependent cytotoxicity of antibodies.
  • the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Starr, Md. (1991). In the present application, amino acid residues in variable domain sequences and full-length antibody sequences were determined using the rules encompassing the Kabat definition (see Table 1).
  • Laa-Lbb can refer to the amino acid sequence from the N-terminus of the antibody light chain, from aa to bb;
  • Haa-Hbb can refer to the amino acid sequence from aa to bb from the N-terminus of the antibody heavy chain sequence.
  • L24-L34 can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from positions 24 to 34;
  • H231-H35 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain, from the 31st to the 35th amino acid sequence.
  • Fab refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested with papain as described above. Papain digestion of the antibody yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region, supra).
  • a Fab fragment consists of a complete L chain with the variable region of a heavy chain and the first constant region (C H 1 ) of the H chain (V H ).
  • Fab' fragment refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment.
  • a Fab' fragment includes all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain.
  • Fab' fragments may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 refers to antibody fragments produced by pepsin digestion of whole antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen-binding activity and are capable of cross-linking antigens.
  • Fv fragment refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions.
  • Heavy chain variable regions and light chain variable regions include, for example, CDRs.
  • Fv fragments include all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains can be The variable regions are contiguous (eg, via synthetic linkers such as short flexible polypeptide linkers) and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a scFv may have the VL and VH variable regions described in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include a VL-linker-VH Or VH-linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain, a VL domain, or a VH domain or a VL domain, see eg Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible minor natural mutations.
  • Monoclonal antibodies are usually highly specific for a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • the term "reference antibody” generally refers to any antibody that can bind to an antigen (eg, PD-1).
  • the antigen binding proteins described herein can compete with a reference antibody for binding to an antigen (eg, PD-1).
  • isolated nucleic acid molecule or isolated polynucleotide
  • isolated nucleic acid molecule generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, which is not related to the polynucleus found in nature All or a portion of the nucleotides are associated, or linked, to polynucleotides to which they are not linked in nature.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the term "cell” generally refers to an individual cell, cell line or cell that can or already contains a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. cell culture.
  • the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be mammalian cells.
  • the mammalian cells can be 293T cells.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector has been introduced.
  • the recombinant host cells include not only certain specific cells, but also progeny of these cells.
  • the term "pharmaceutically acceptable adjuvant” generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are free of the cells or mammals to which they are exposed at the doses and concentrations employed. poisonous.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, Sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be a substitution, deletion or addition of one or more in the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the PD-1 protein).
  • a protein or polypeptide of multiple amino acids may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • the homologue may be at least about 85% of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the PD-1 protein) (eg, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) Proteins or polypeptides with sequence homology.
  • the polypeptide eg, an antibody or fragment thereof that specifically binds to the PD-1 protein
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • FASTA and BLAST A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Protein Similarity Search", Science, 227: 1435-1441, 1989.
  • a description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215: 403-410 , 1990.
  • the terms "about” and “approximately” shall generally mean an acceptable degree of error in the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%) of a given value or range of values, generally within 10% thereof, and more generally within 5% thereof.
  • the term "therapeutically effective amount” refers to the amount of an antibody that, when administered to a human or animal, elicits a response sufficient to produce a therapeutic effect in said human or animal.
  • An effective amount can be readily determined by one of ordinary skill in the art according to routine methods.
  • the present application provides an antigen-binding protein, which may comprise at least one CDR in the VH of an antibody heavy chain variable region, the VH comprising the amino acid sequence shown in SEQ ID NO: 7 or 9.
  • Antigen-binding proteins described herein include antibodies or antigen-binding fragments thereof.
  • the antibodies described herein can be monoclonal antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies.
  • Antigen-binding fragments of the antibodies described herein may be Fab, Fab', Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAbs.
  • the antigen binding protein described in this application can compete with the reference antibody for binding to PD-1.
  • the reference antibody may comprise a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5;
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6;
  • the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1;
  • the The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1.
  • the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.
  • antigen binding proteins described herein may also comprise heavy chain framework regions HFR1, HFR2, HFR3 and HFR4.
  • the HFR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 19, and the C-terminus of the HFR1 is directly or indirectly linked to the N-terminus of the HCDR1.
  • the HFR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 20, and the HFR2 is located between the HCDR1 and the HCDR2.
  • the HFR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 21, and the HFR3 is located between the HCDR2 and the HCDR3.
  • the HFR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 22, and the N-terminus of the HFR4 is linked to the C-terminus of the HCDR3.
  • the antigen binding protein may comprise an antibody heavy chain variable region (VH), and the VH may comprise the amino acid sequence shown in SEQ ID NO: 7 or 9.
  • VH antibody heavy chain variable region
  • HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may comprise the amino acids shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, in order sequence.
  • the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 can respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 in turn.
  • HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may comprise the amino acids shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 in sequence, respectively sequence.
  • the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 can respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 in turn.
  • the antigen binding proteins described herein may comprise heavy chain constant regions.
  • the heavy chain constant region may comprise the constant region of human IgG.
  • the heavy chain constant region can include a human IgG4 constant region and/or a human IgG1 heavy chain constant region.
  • the heavy chain constant region may comprise the amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.
  • the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in sequence, respectively, and the heavy chain of the antigen binding protein is constant
  • a region may comprise the amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.
  • the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 may respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the heavy chain constant region of the antigen-binding protein may comprise The amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
  • the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 may respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the heavy chain constant region of the antigen-binding protein may comprise The amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.
  • the antigen binding protein described in the present application may comprise at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence shown in SEQ ID NO: 8 or 10.
  • the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.
  • the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in order, and the antigen binding protein Can comprise at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO:8.
  • the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in order, and the antigen binding protein Can comprise at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO:16.
  • the antigen binding proteins described herein may comprise light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.
  • the LCDR1 of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:4.
  • the LCDR2 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:5.
  • the LCDR3 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:6.
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in sequence.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO: 1, The amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:7.
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:9.
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:7
  • the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise any of SEQ ID NO:27 or SEQ ID NO:28 An amino acid sequence shown.
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:9
  • the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise any of SEQ ID NO:27 or SEQ ID NO:28 An amino acid sequence shown.
  • the antigen binding proteins described herein may comprise light chain framework regions LFR1, LFR2, LFR3 and LFR4.
  • the LFR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 23, and the C-terminus of the LFR1 is directly or indirectly linked to the N-terminus of the LCDR1.
  • the LFR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 24, and the LFR2 is located between the LCDR1 and the LCDR2.
  • the LFR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 25, and the LFR3 is located between the LCDR2 and the LCDR3.
  • the LFR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 26, and the N-terminus of the LFR4 is linked to the C-terminus of the LCDR3.
  • the antigen binding protein may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 8 or 10.
  • the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 in sequence .
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in sequence
  • LFR1, LFR2 of the antigen binding protein , LFR3 and LFR4 in turn may comprise the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
  • HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise SEQ ID NO: 15.
  • the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, in sequence .
  • LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in sequence
  • LFR1, LFR2 of the antigen binding protein , LFR3 and LFR4 in turn may comprise the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
  • HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise SEQ ID NO: 23.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 7 or 15
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 8 or 10 amino acid sequence.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:7
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:8.
  • HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively
  • LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn
  • HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: : 11, SEQ ID NO: 12, SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14, and the LFR1, LFR2, LFR3 and LFR4 of the antigen-binding protein may comprise SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: 14 in turn
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:9
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:10.
  • HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively
  • LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn
  • HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: Amino acid sequences shown in : 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.
  • the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 in turn.
  • the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise the amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:7
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:8, and the heavy chain constant region of the antigen binding protein The amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28 may be included.
  • HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively
  • LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn
  • HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: : 11, SEQ ID NO: 12, SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14, and the LFR1, LFR2, LFR3 and LFR4 of the antigen-binding protein may comprise SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: 14 in turn ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 7, and the VL may comprise SEQ ID NO: 8
  • the VH can comprise the amino acid sequence set forth in SEQ ID NO:9
  • the VL can comprise the amino acid sequence set forth in SEQ ID NO:10.
  • HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively
  • LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn
  • HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO:
  • the amino acid sequences shown in: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, and the LFR1, LFR2, LFR3 and LFR4 of the antigen-binding protein may comprise SEQ ID NO: 23, SEQ ID NO: 23, SEQ ID NO: 22 in turn.
  • SEQ ID NO: 24 SEQ ID NO: 25, and SEQ ID NO: 26, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 15, and the VL may comprise SEQ ID NO: 16
  • the amino acid sequence shown, and the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
  • the physical/chemical properties and/or biological activities of the PD-1 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.
  • the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like Antigen-binding activity of an antigen-binding protein or fusion protein.
  • Antigen binding proteins eg, PD-1 antibodies
  • Antigen binding proteins eg, PD-1 antibodies
  • Antigen-binding proteins eg, PD-1 antibodies
  • Antigen-binding proteins that "specifically bind" the PD-1 antigen can generally bind PD-1, but not other proteins that lack the PD-1 sequence.
  • Antigen-binding proteins eg, PD-1 antibodies
  • PD-1 antibodies are capable of specifically binding PD-1 antigen or a labeled form thereof (eg, fluorescently labeled PD-1 antigen), but not binding epitopes lacking PD-1 of other proteins.
  • an antigen binding protein eg, an antibody
  • binds to a PD-1 antigen can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include Biofilm Interferometry (BLI).
  • the antigen binding proteins described in this application can bind to human PD-1 protein.
  • the antigen binding proteins described herein can also cross-react with PD-1 of monkeys (eg, cynomolgus monkeys). For example, detected by flow cytometry and ELISA.
  • cross-reactivity refers to the ability of an antibody to react with homologous proteins from other species.
  • the binding activity of the antigen-binding proteins described herein to PD-1 can be detected using an enzyme-linked immunosorbent assay.
  • the PD-1 antigen binding protein in an ELISA using human PD-1 antigen protein, can have an EC50 value for PD-1 between about 0.0001 nM to about 100 nM, eg, about 0.001 nM to about 10 nM between, can be between about 0.001 nM and about 5 nM, can be between about 0.001 nM and about 1 nM, or can be between about 0.01 nM and about 1 nM.
  • the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1.
  • the antigen binding protein blocks the binding of PD-1 to PD-L1 in an ELISA assay. For example, first, the PD-L1 antigen protein is coated on the plate, and the unlabeled antigen-binding protein and the biotin-labeled PD-1 protein are mixed with decreasing amounts, and then incubated together. Cells were then analyzed using ELISA to confirm that the antigen binding protein could block the binding of PD-1 and PD-L1.
  • the antigen binding protein described in the present application can stimulate the proportion of CD07a-expressing cells in immune cells.
  • the antigen binding proteins described in the present application can stimulate the secretion of IFN- ⁇ , TNF- ⁇ and/or IL2 in immune cells.
  • the stimulating ability of the antigen binding protein described in the present application can be based on the activation of known activators in the art (such as CD3 antibody, CD28 antibody, or nanomaterial TransAct TM comprising CD3 antibody and CD28 antibody), increasing the The effect of immune cell activation.
  • the immune cells may include lymphocytes such as B cells, T cells, natural killer cells, myeloid cells such as monocytes, macrophages, mast cells, basophils and granulocytes.
  • the immune cells may comprise tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • the immune cells may comprise artificially modified immune cells.
  • the immune cells may comprise TCR-T cells.
  • the secretion of cytokines from immune cells can be measured by methods known to any person skilled in the art, for example, by enzyme-linked immunosorbent assay (ELISA) to quantify the proliferation of immune cells (eg, T cells) or cytokines produced by immune cells (eg, by enzyme-linked immunosorbent assay). IFN- ⁇ or IL-2 produced by T cells).
  • ELISA enzyme-linked immunosorbent assay
  • IFN- ⁇ or IL-2 produced by T cells.
  • the stimulation results of PD-1 antibody on T lymphocytes can be detected by mixed lymphocyte reaction (MLR) assay.
  • MLR mixed lymphocyte reaction
  • the application provides one or more nucleic acid molecules that can encode the isolated antigen binding proteins described herein.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the present application provides a vector, which can comprise the nucleic acid molecule described herein.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host. Such control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the present application provides a cell, which may comprise the nucleic acid molecule described herein or the vector described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the application provides a pharmaceutical composition, which can comprise the antigen binding protein and/or the nucleic acid molecule described herein, the carrier, the host cell, and optionally a pharmaceutical acceptable adjuvants.
  • the pharmaceutically acceptable adjuvants are not toxic to recipients at the doses and concentrations employed, and may include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, pro- Aqueous polymers, amino acids, carbohydrates, salt-forming counterions, metal complexes, and/or nonionic surfactants.
  • the pharmaceutical compositions herein may also contain more than one active compound, typically those active compounds with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.
  • compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antigen binding protein.
  • the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
  • the present application provides methods for preparing the antigen binding proteins.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application.
  • an animal can be immunized with a linked or naturally occurring PD-1 protein or fragment thereof.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • PD-1 can be used as an immunogen (antigen) for generating non-human antibodies specific for PD-1 and screening the antibodies for biological activity.
  • the priming immunogen can be full-length mature human PD-1, including native homodimers, or peptides containing single/multiple epitopes.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE.
  • the antibody is an IgG antibody, and the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below.
  • any type of light chain can be used in the compounds and methods of the present application. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present application.
  • sequences of the DNA molecules of the antigen-binding proteins or fragments thereof of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the nucleic acid molecule can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present application also relates to vectors comprising suitable nucleic acid molecules as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • animal cells can include (but are not limited to): 293T cells.
  • the steps of transforming host cells with recombinant DNA described in this application can be performed using techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultured with conventional media under appropriate conditions. Typically, the transformed host cells are cultured under conditions suitable for expression of the antigen binding proteins of the present application.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. skilled in the art
  • the antigen-binding protein of the present application can be obtained by conventional separation and purification methods well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as flow cytometric sorting (FACS) or enzyme-linked immunosorbent assay (ELISA).
  • FACS flow cytometric sorting
  • ELISA enzyme-linked immunosorbent assay
  • the present application provides a use of the antigen-binding protein in the preparation of a medicine.
  • the antigen binding protein can be administered alone or in combination with one or more additional therapies, such as chemoradiation therapy, immunotherapy, surgical intervention, or any combination of these.
  • the medicament can be used to treat a PD-1 mediated disease or disorder, eg, to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the antigen-binding proteins provided in the present application can be used to prevent or treat diseases or conditions mediated by PD-1.
  • the preventing or treating a disease or disorder may refer to inhibiting or delaying the development or progression of the disease or disorder.
  • it can be used to inhibit the development or progression of tumors.
  • tumor growth or tumor cell proliferation can be inhibited.
  • the present application provides said antigen binding proteins for use in the prevention or treatment of PD-1 mediated diseases or disorders.
  • the present application provides a method of inhibiting the binding of PD-1 to PD-L1, comprising administering the antigen binding proteins described herein.
  • the method can be an ex vivo or in vitro method.
  • the method can include contacting a biological sample with an antigen binding protein and/or PD-L1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind PD-L1 , detecting whether a complex is formed between the antigen binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1.
  • the present application provides a method of preventing, alleviating or treating a PD-1 mediated disease or condition, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof described herein, the Molecular nucleic acid, the vector, the host cell and/or the pharmaceutical composition.
  • a method of preventing, alleviating or treating a PD-1 mediated disease or condition comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof described herein, the Molecular nucleic acid, the vector, the host cell and/or the pharmaceutical composition.
  • it can be used to inhibit the development or progression of tumors.
  • tumor growth or tumor cell proliferation can be inhibited.
  • Example 1 Expression vector construction and eukaryotic expression of the fusion protein (PD-1-huIgG1 Fc) of recombinant human programmed death receptor 1 (PD-1) amino acid sequence 25 to 167 and human IgG1 Fc region
  • the gene sequence from the 25th isoleucine to the 167th glutamine of the programmed death receptor 1 (PD-1) (NCBI accession No.NP_005009.2) was synthesized by chemical synthesis (SEQ ID NO: 29), the gene sequence encodes the amino acid sequence shown in SEQ ID NO: 30.
  • the gene sequence from the 100th proline to the 330th lysine in the constant region of the human IgG1 heavy chain was synthesized by chemical synthesis.
  • Chemically synthesized upstream primers containing mouse Igkv3-10 signal peptide gene sequence were used for expression vector construction.
  • the PD-1 gene fragment was spliced with the human IgG1 Fc gene fragment.
  • the spliced product was cloned into pCDNA3.1 (Thermo) using the TaKaRa seamless cloning kit.
  • the culture supernatant was collected, and the recombinant PD-1-huIgG1 Fc fusion protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant PD-1-huIgG1 Fc fusion protein was shown to be about 60k Daltons in size by Coomassie brilliant blue staining after reducing SDS-PAGE electrophoresis.
  • Example 2 Construction of expression vector and eukaryotic expression of recombinant human programmed death receptor 1 (PD-1) amino acid sequence 25 to 167 and polyhistidine tag fusion protein (PD-1-his)
  • PD-1-his human programmed death receptor 1
  • the gene sequence from the 25th isoleucine to the 167th glutamine of programmed death receptor 1 (PD-1) (NCBI accession No. NP_005009.2) was synthesized by chemical synthesis.
  • the upstream primer containing the mouse Igkv3-10 signal peptide gene sequence and the downstream primer containing the polyhistidine tag (having the amino acid sequence shown in SEQ ID NO:32) gene sequence (SEQ ID NO:31) were chemically synthesized for The pCDNA3.1 vector expressing PD-1-his was constructed.
  • the culture supernatant was collected, and the recombinant PD-1-his protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant PD-1-his protein was shown to be about 40k Daltons in size by Coomassie brilliant blue staining after reducing SDS-PAGE electrophoresis.
  • Example 3 Construction of anti-human PD-1 antibody (Nivolumab) expression vector and eukaryotic expression
  • the light and heavy chain variable region genes of Nivolumab antibody were synthesized by chemical synthesis as SEQ ID NO: 33 and SEQ ID NO: 34, respectively.
  • the light chain variable region of Nivolumab antibody has the amino acid sequence shown in SEQ ID NO: 35.
  • Nivolumab antibody heavy The chain variable region has the amino acid sequence shown in SEQ ID NO:36.
  • the heavy chain variable region gene was amplified by PCR, and the amplified product was cloned into pFUSEss containing human IL-2 signal peptide gene and human IgG1 heavy chain constant region gene using TaKaRa seamless cloning kit. -CHIg-hG1 (invivogen).
  • the light chain variable region fragment was amplified by PCR, and the amplified product was cloned into a human IL-2 signal peptide gene and human kappa light chain constant region gene using the TaKaRa seamless cloning kit.
  • pFUSE2ss-CLIg-hK invivogen.
  • the culture supernatant was collected, and the Nivolumab antibody was purified with AKTA explorer 100 (GE).
  • the Nivolumab antibody was shown to be approximately 150 k Daltons in size by Coomassie brilliant blue staining after non-reducing SDS-PAGE electrophoresis.
  • Example 4 ELISA detection of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) binding to Nivolumab antibody
  • Enzyme-linked immunosorbent assay was used to detect the binding of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) to Nivolumab antibody.
  • PD-1-huIgG1 Fc or PD-1-his fusion protein 100ng/well, coated overnight at 4°C.
  • PBS was washed three times, 1% BSA/PBS was added, 200uL/well, and the cells were blocked at 37°C for 1 hour. After washing the plate with 100ul PBS, add 100ng/well of Nivolumab chimeric antibody, and bind at 37°C for 1 hour.
  • Example 5 Expression of tetravalent human programmed cell death-ligand 1 (PD-L1) extracellular segment mutants and determination of their affinity with human PD-1 molecules
  • the gene sequence (SEQ ID NO:37) of two human PD-L1 extracellular segment mutants linked by a flexible peptide was synthesized by chemical synthesis, and the gene sequence encoded the amino acid sequence shown in SEQ ID NO:38.
  • the gene sequence from the 100th proline to the 330th lysine of the human IgG1 heavy chain constant region was synthesized by chemical synthesis.
  • Chemically synthesized upstream primers containing mouse Igkv3-10 signal peptide gene sequence were used for expression vector construction.
  • the gene fragment of the PD-L1 extracellular segment mutant repeat structure was spliced with the human IgG1 Fc gene fragment.
  • the spliced product was cloned into pCDNA3.1 (Thermo) using the TaKaRa seamless cloning kit.
  • the culture supernatant was collected, and the tetravalent human PD-L1 extracellular segment mutant protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the tetravalent human PD-L1 extracellular segment mutant protein was shown to be about 85k Daltons in size by Coomassie brilliant blue staining after reducing SDS-PAGE electrophoresis.
  • PD-1-huIgG1 Fc fusion protein was randomly biotinylated using standard operating procedures provided by EZ-Link Sulfo-NHS-LC-Biotin (Thermo). The binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA.
  • the affinity of tetravalent human PD-L1 extracellular segment mutants to PD-1-huIgG1 Fc was determined using Octet K2 (ForteBio) molecular interaction analyzer. Following the standard operating procedure of Octet K2, 100 nM of biotinylated PD-1-huIgG1 Fc fusion protein was prepared and immobilized on SA probe (ForteBio) at a height of 1 nM.
  • the tetravalent human PD-L1 ecto-segment mutant was used as the analyte in a two-fold serial dilution starting from a concentration of 50 nM, and the affinity of the quadrivalent human PD-L1 ecto-segment mutant with PD-1-huIgG1 Fc was determined.
  • the resultant force was 9.4 nM and the results of the affinity assay are shown in FIG. 1 .
  • Wild-type PD-1 and PD-L1 have a known binding affinity of 8.2 uM (see Cheng, X. et al., J. Biol. Chem., 288(17):11771-85, 2013).
  • mice were sacrificed four days after the direct intraperitoneal injection with PD-1-huIgG1 Fc fusion protein for shock immunization, and the spleen was removed.
  • Spleen cells were obtained by triturating the whole spleen with a 70 micron cell mesh (BD). After washing twice with PBS, spleen cells were obtained by centrifugation at 1000g for 10 minutes. Total RNA was extracted using Trizol RNA extraction kit.
  • the first-strand cDNA was synthesized using the SuperScript TM IV First-Strand Synthesis System kit.
  • PCR reaction was performed as follows: pre-denaturation at 98°C for 1 minute followed by temperature cycling, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
  • the amplified VH and VL genes were recovered using a DNA gel recovery kit.
  • the scFv gene was amplified by overlapping PCR using the upstream primer scFv-F and the downstream primer scFv-R after mixing the same amount of VH gene and VL gene as a template.
  • the 50uL reaction system add 25uL phusion master mix, 2.5uL (25pmol) upstream primer, 2.5uL (25pmol) downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O.
  • the PCR reaction was performed as follows: pre-denaturation at 98°C for 1 minute followed by temperature cycling, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
  • the amplified scFv gene fragments were recovered using a DNA gel recovery kit.
  • the scFv gene fragment and pcomb3XTT vector were digested with SfiI endoDNA, respectively.
  • the digested scFv gene fragment and pcomb3XTT vector were recovered using DNA gel recovery kit.
  • the scFv gene fragment and the digested pcomb3XTT vector were digested with T4 ligase cyclase.
  • T4 ligase cyclase In a 50uL reaction system, add 1uL of T4 ligase, 5uL of 10x buffer, 100ng of scFv gene, 500ng of pComb3XTT vector, and add ddH2O to 50uL. After thorough mixing, incubate at 4°C for 16 hours. Take a small amount of product to verify the ligation efficiency by agarose gel electrophoresis.
  • PD-1-huIgG1 Fc fusion protein was randomly biotinylated using standard operating procedures provided by EZ-Link Sulfo-NHS-LC-Biotin. The binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA.
  • PD-1-huIgG1 Fc fusion protein As the target protein, biopanning was performed, and the above-mentioned murine immune antibody library was biopanned to obtain antibodies that bind to the PD-1-huIgG1 Fc fusion protein (especially the extracellular domain of PD-1). .
  • the phage was precipitated with PEG/NaCl, and the phage pellet was dissolved with glycerol/PBST to obtain a phage suspension of the immune library.
  • the casein-blocked phage was put into the co-incubation system of casein-blocked biotinylated huIgG1 Fc (ACRO Biosystems) fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the supernatant phage suspension was collected.
  • the collected phage suspension was put into the co-incubation system of casein-blocked biotinylated PD-1-huIgG1 Fc fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the magnetic beads were washed with PBST. Phages that cannot bind to the PD-1-huIgG1 Fc fusion protein are removed. Under appropriate elution conditions, the phage was subjected to competitive elution using a homemade tetravalent PD-L1 ecto-segment mutant.
  • the antibody library obtained after the third round of biopanning was diluted and spread on a plate containing ampicillin to obtain monoclones, and the monoclones were selected and cultured overnight in deep-well plates. The next day, the deep-well plate was freeze-thawed three times, and the supernatant was centrifuged for the subsequent two types of ELISA reactions.
  • ELISA reaction to detect binding activity coat with 50ng PD-1-his overnight, wash with PBS three times, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, 100ul of the centrifuged supernatant was added and incubated at 37°C for 1 hour. After washing three times with PBST, 100ul of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added at a 1:5000 dilution.
  • ELISA reaction for detecting blocking activity coat with PD-1-his overnight, wash with PBS three times, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, add 100ul of centrifugal supernatant and 20ng of tetravalent PD-L1 mixture and incubate at 37°C for 1 hour. After washing three times with PBST, add 100ul of 1:5000 diluted HRP-conjugated goat anti-mouse IgG (Fab specific) (Thermo).
  • candidate clones that meet the above two conditions at the same time are sequenced to obtain the antibody light chain variable domain and heavy chain variable domain sequences.
  • the candidate clones obtained from the primary screening were expanded and cultured with a 50ml shaker tube (thermo), and the periplasmic cavity extract was obtained by lysozyme (Shanghai Shenggong) combined with three freeze-thaw methods, and two ELISAs were used again to identify the binding and blocking activity. Coat with 50ng PD-1-his overnight, wash three times with PBS, add 1% BSA/PBS, 200uL/well, and block for 1 hour at 37°C. After washing three times with PBS, 100ul of the centrifuged supernatant was added and incubated at 37°C for 1 hour.
  • the heavy chain variable region sequences of 6H6 were cloned into pFUSEss-CHIg-hG1, respectively.
  • the 6H6 light chain variable region sequences were cloned into pFUSE2ss-CLIg-hK, respectively.
  • 293T cells ATCC
  • ATCC ATCC
  • 293T cells ATCC
  • the 6H6 antibody was purified with AKTA explorer 100 (GE).
  • the 6H6 antibody was shown to be approximately 150 k Daltons in size by Coomassie brilliant blue staining after non-reducing SDS-PAGE electrophoresis.
  • 6H6, Nivolumab and hIgG1 isotype control were used for competition ELISA experiments.
  • the brief steps are: overnight coating with 100ng of PD-1-huIgG1 Fc fusion protein per well, followed by adding 50ul 2ng/ul of different hIgG1 antibodies (6H6, Nivolumab and isotype control antibody) and 50ul of PD-L1- His (ACRO Biosystems) mixture, 100ul 1:5000 mouse anti-his tag-HRP (Thermo) color development to detect the blocking effect of each antibody molecule level.
  • This detection step contains three duplicate wells, and the average value of ELISA results is shown in Table 3.
  • the molecular level blocking effect of 6H6 is better than that of Nivolumab.
  • the residues are close to the CDR region and are embedded in the interior of the protein.
  • the third type of amino acid residues are residues that have direct interactions with the CDR region, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Based on the above rules, three candidate molecules were constructed, and the Hu_6H6 molecule was selected as the humanized 6H6 antibody version by in vitro affinity determination.
  • the amino acid sequences of the antibody heavy chain variable region and light chain variable region of Hu_6H6 were sequence No. 15 and 1, respectively. NO.16.
  • the plate was washed 3 times with 0.1% PBST, patted dry, and then incubated with a mixture of scFv-huIgG1 Fc-type antibody and Biotinylated PD-1-huIgG1 Fc (Kactus Biosystems) for 1 hour at room temperature (2ug/ml 50ul of PD-1-huIgG1 Fc with 160ug /ml initial two-fold serial dilution of 50ul antibody mix, a total of eight gradients).
  • the plate was washed three times with 0.1% PBST, and 100ul of Streptavidin-HRP (R&D Systems) was added to each well (1:200 dilution) and incubated at room temperature for 1 hour.
  • ELISA was used to determine the affinity of PD-1 antibody to verify the affinity of huIgG1 antibody of 6H6 and pembrolizumab: four times overnight coating PD-1 Protein, Human, Recombinant (His Tag) (Beijing Yiqiao Shenzhou) 0.5ug/ml 100ul per well , wash the plate and pat dry, then use 1% BSA in PBS 200ul/well to incubate for 1 hour at 37°C for blocking.
  • the plate was washed 3 times with 0.1% PBST, patted dry, and then incubated with scFv-huIgG1 Fc-type antibody for 1 hour at room temperature (two-fold serial dilution starting from 10ug/ml, a total of eight gradients). Wash the plate 3 times with 0.1% PBST, add Goat anti-Mouse IgG F(ab')2Secondary Antibody,HRP(Thermo)/Goat Anti-Human IgG Secondary Antibody(HRP)(Beijing Yiqiao Shenzhou) 100ul per well (1:10000 dilution) for 1 hour at room temperature.
  • TILs tumor-infiltrating lymphocytes
  • TIL tumor infiltrating lymphocytes
  • Donor A and Donor B Two batches of TIL cells (Donor A and Donor B) were resuscitated and cultured in IL-2-containing medium for 48 hours. After the culture, IL-2 was removed by washing, and 1E5/well cells were added to 96-well plates.
  • the experimental group (Transact+PD-1) was added with T Cell TransAct TM (Miltenyi Biotec) and 1 ⁇ g according to the instructions at a ratio of 1:1000. /ml 6H6 PD-1 antibody stimulation, the control group only added T Cell TransAct TM , after overnight incubation, the proportion of cells expressing CD107a and the results of cytokine secretion were detected.
  • Figure 4A shows the results of the proportion of CD107a cells added to PD-1 antibody in the tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A.
  • Figure 4B shows the results of the proportion of CD107a cells added to the PD-1 antibody in the tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor B. The results show that the PD-1 antibody of the present application can increase the proportion of cells expressing CD107a.
  • TIL tumor-infiltrating lymphocyte
  • FIGS 5A-5F show the cytokines IL-2, TNF- ⁇ and/or IFN- ⁇ added to PD-1 antibody in tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B Secretion results graph.
  • TIL tumor-infiltrating lymphocyte
  • Example 9 Mixed lymphocyte reaction (MLR) assay to detect PD-1 antibody to stimulate T lymphocytes
  • the mixed lymphocyte reaction (MLR) assay was used to detect that PD-1 antibody could stimulate T lymphocytes to secrete IL-2 and IFN- ⁇ .
  • the cell density of human PBMC was adjusted to 2E7 cells/ml, and the monocytes were sorted by CD14 MicroBeads, human (Miltenyi Biotec). 50ng/ml GM-CSF (ACRO Biosystems) and 50ng/ml IL-4 (ACRO Biosystems) were added and cultured for 5 days.
  • DC cell maturation was induced by supplementation of 10 ng/ml of LPS (Sigma) and 20 ng/ml of TNF- ⁇ (ACRO Biosystems).
  • CD4+ T cells were isolated from PBMCs of various human origins using Naive CD4+ T Cell Isolation Kit II, human (Miltenyi Biotec).
  • 250 ⁇ l of culture medium per well contains 1.0E5 isolated T cells, 2E4 induced mature DC cells, and a series of concentration gradients of pembrolizumab or the PD-1 antibody 6H6 of the present application.
  • a Human IgGl Isotype control was used as a negative control.
  • the mixed lymphocytes were cultured in a 37°C, 5% CO2 cell incubator for 3 days, and then 100ul of the culture supernatant was removed from each well of the 96-well plate for IL-2 and IFN- ⁇ assays.
  • Figures 6A-6B show the results of the mixed lymphocyte reaction (MLR) assay to detect the stimulation results of PD-1 antibody on the secretion of IL-2 and IFN- ⁇ by T lymphocytes.
  • MLR mixed lymphocyte reaction
  • Example 10 PD-1 antibody enhances the killing ability of immune cells
  • TCR-T cells were sorted from freshly isolated human PBMC cells by CD3 MicroBeads, human (Miltenyi Biotec 130-050-101).
  • TCR-T cells can be obtained by any transduction method, for example, lentivirus carrying NY-ESO-1TCR (Kite Pharma) is used to transduce the sorted CD3-positive T cells, and the transduction efficiency can be 40% by flow detection .
  • A375 cells were plated in 96-well plates with 2E4 cells per well, NYESO-1TCR cells transduced with 2E4/well were added, and 10 ⁇ g/ml of 6H6 antibody and control antibody pembrolizumab were added as the experimental group, and the same amount of PBMC cells were added to the A375 cells As a negative control, only A375 cells were used as a blank control, and the fluorescent dye SuperView TM 488 Caspase-3 was added to each well of all groups at the same time. The 96-well plate was transferred to IncuCyte S3 every 3 hours, and pictures were taken automatically for 33 hours, and the killing results were statistically analyzed after 33 hours.
  • Figure 7 shows the results of the enhanced killing ability of TCR-T cells by adding PD-1 antibody.
  • the results show that the PD-1 antibody of the present application can enhance the killing ability of immune cells.

Abstract

Provided is an antigen-binding protein capable of binding to PD-1. Also provided are a preparation method for and an application of the antigen-binding protein.

Description

PD-1抗体及其制备方法与应用PD-1 antibody and its preparation method and application 技术领域technical field
本申请涉及生物医药领域，具体的涉及一种结合PD-1的抗原结合蛋白。The present application relates to the field of biomedicine, in particular to an antigen-binding protein that binds to PD-1.
背景技术Background technique
程序性死亡受体1(Programmed Death-1,PD-1)是具有288个氨基酸的I型膜蛋白，主要表达在激活的T细胞表面。PD-1有两个配体，即程序性死亡配体-1(Programmed Death Ligand-1，PD-L1)和PD-L2。PD-1与PD-L1及PD-L2相互作用会下调T细胞的活性，减弱细胞因子的分泌，起到免疫抑制作用。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，发挥杀伤肿瘤细胞的作用，进而抑制肿瘤生长。因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。目前，PD-1/PD-L1通路的阻断型抗体药物在临床上仍面临诸多挑战，例如有效性低、耐药性和副作用等，仍有必要继续开发出更有效的抗PD-1抗体。Programmed death-1 (PD-1) is a type I membrane protein with 288 amino acids, mainly expressed on the surface of activated T cells. PD-1 has two ligands, namely programmed death ligand-1 (Programmed Death Ligand-1, PD-L1) and PD-L2. The interaction of PD-1 with PD-L1 and PD-L2 can down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive effect. PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, restore the activity of T cells, and play a role in killing tumor cells, thereby inhibiting tumor growth. Therefore, immunomodulation targeting PD-1/PD-L1 has important implications for tumor suppression. At present, anti-PD-1/PD-L1 pathway blocking antibody drugs still face many clinical challenges, such as low efficacy, drug resistance and side effects, and it is still necessary to continue to develop more effective anti-PD-1 antibodies .
发明内容SUMMARY OF THE INVENTION
本申请提供了一种结合PD-1的抗原结合蛋白，其表现出一种或多种期望的功能特性，例如与PD-L1的高亲和力结合、抑制PD-1与PD-1结合的能力、增强T细胞活化包括增殖能力、IFN-γ和/或IL-2分泌的能力、刺激抗体应答的能力和/或逆转免疫抑制细胞如T调节细胞的抑制功能的能力。在一个实施方式中，本申请还提供了一种scFv-huIgG1 Fc型抗体，方便以自分泌形式与细胞药物联合使用。本申请还提供编码所述的分离的抗原结合蛋白的核酸分子、表达载体、宿主细胞和用于制备所述的分离的抗原结合蛋白的方法。本申请公开的结合PD-1的抗原结合蛋白可以用于(单独或结合其他活性剂或治疗形式)治疗、预防和/或诊断疾病，如癌症疾病(例如，实体和软组织肿瘤)。The present application provides an antigen-binding protein that binds PD-1, which exhibits one or more desired functional properties, such as high-affinity binding to PD-L1, the ability to inhibit the binding of PD-1 to PD-1, Enhanced T cell activation includes the ability to proliferate, IFN-γ and/or IL-2 secretion, ability to stimulate antibody responses, and/or ability to reverse the suppressive function of immunosuppressive cells such as T regulatory cells. In one embodiment, the present application also provides a scFv-huIgG1 Fc-type antibody, which is convenient to be used in combination with cellular drugs in an autocrine form. The application also provides nucleic acid molecules encoding the isolated antigen binding proteins, expression vectors, host cells and methods for preparing the isolated antigen binding proteins. Antigen binding proteins that bind PD-1 disclosed herein can be used (alone or in combination with other active agents or therapeutic modalities) to treat, prevent and/or diagnose diseases, such as cancer diseases (eg, solid and soft tissue tumors).
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:7或9所示的氨基酸序列。In one aspect, the application provides an isolated antigen-binding protein comprising at least one CDR in the variable VH of an antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO: 7 or 9.
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体或其抗原结合片段。In certain embodiments, the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb。In certain embodiments, the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and/or dAb.
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。In certain embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
在某些实施方式中，所述的分离的抗原结合蛋白与参比抗体竞争结合PD-1，其中所述参 比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列；所述LCDR2包含SEQ ID NO:5所示的氨基酸序列；所述LCDR3包含SEQ ID NO:6所示的氨基酸序列，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2包含SEQ ID NO:2所示的氨基酸序列；所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein competes with a reference antibody for binding to PD-1, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, and the reference antibody has a The light chain variable region comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:5: The amino acid sequence shown in 6, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence shown; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
在某些实施方式中，所述的分离的抗原结合蛋白的所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。In certain embodiments, the VH of the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3.
在某些实施方式中，所述的分离的抗原结合蛋白的所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。In certain embodiments, the HCDR2 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:2.
在某些实施方式中，所述的分离的抗原结合蛋白的所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。In certain embodiments, the HCDR1 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:1.
在某些实施方式中，所述的分离的抗原结合蛋白的所述VH包含FR1，FR2和FR3，FR4，其中所述FR1包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列。In certain embodiments, the VH of the isolated antigen binding protein comprises FR1, FR2 and FR3, FR4, wherein the FR1 comprises the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:19.
在某些实施方式中，所述的分离的抗原结合蛋白的所述FR2包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列。In certain embodiments, the FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:20.
在某些实施方式中，所述的分离的抗原结合蛋白的所述FR3包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列。In certain embodiments, the FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:13 or SEQ ID NO:21.
在某些实施方式中，所述的分离的抗原结合蛋白的所述FR4包含SEQ ID NO:14或SEQ ID NO:22所示的氨基酸序列。In certain embodiments, the FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:14 or SEQ ID NO:22.
在某些实施方式中，所述的分离的抗原结合蛋白包含VH，且所述VH包含SEQ ID NO:7或9所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 7 or 9.
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体重链恒定区。In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.
在某些实施方式中，所述的分离的抗原结合蛋白的所述重链恒定区源自人IgG恒定区。In certain embodiments, the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG constant region.
在某些实施方式中，所述的分离的抗原结合蛋白的所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。In certain embodiments, the heavy chain constant region of the isolated antigen binding protein is derived from a human IgG4 constant region and/or a human IgGl constant region.
在某些实施方式中，所述的分离的抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:8或10所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises at least one CDR in the variable region VL of an antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10.
在某些实施方式中，所述的分离的抗原结合蛋白的所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:4所示的氨基酸序列。In certain embodiments, the VL of the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4.
在某些实施方式中，所述的分离的抗原结合蛋白的所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。In certain embodiments, the LCDR2 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:5.
在某些实施方式中，所述的分离的抗原结合蛋白的所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。In certain embodiments, the LCDR3 of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:6.
在某些实施方式中，所述的分离的抗原结合蛋白的所述VL包含FR1，FR2和FR3，FR4，其中所述FR1包含SEQ ID NO:15或SEQ ID NO:23所示的氨基酸序列。In certain embodiments, the VL of the isolated antigen binding protein comprises FR1, FR2 and FR3, FR4, wherein the FR1 comprises the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:23.
在某些实施方式中，所述的分离的抗原结合蛋白的所述FR2包含SEQ ID NO:16或SEQ ID NO:24所示的氨基酸序列。In certain embodiments, the FR2 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:16 or SEQ ID NO:24.
在某些实施方式中，所述的分离的抗原结合蛋白的所述FR3包含SEQ ID NO:17或SEQ ID NO:25所示的氨基酸序列。In certain embodiments, the FR3 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:25.
在某些实施方式中，所述的分离的抗原结合蛋白的所述FR4包含SEQ ID NO:18或SEQ ID NO:26所示的氨基酸序列。In certain embodiments, the FR4 of the isolated antigen binding protein comprises the amino acid sequence set forth in SEQ ID NO:18 or SEQ ID NO:26.
在某些实施方式中，所述的分离的抗原结合蛋白包含VL，且所述VL包含SEQ ID NO:8或10所示的氨基酸序列。In certain embodiments, the isolated antigen binding protein comprises VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 8 or 10.
在某些实施方式中，所述的分离的抗原结合蛋白具有下述性质中的一种或多种：In certain embodiments, the isolated antigen binding protein has one or more of the following properties:
a)能够结合人的PD-1；a) capable of binding to human PD-1;
b)能够阻断PD-1和PD-L1结合；b) Can block the binding of PD-1 and PD-L1;
c)能够阻断PD-1和PD-L2结合；c) can block the binding of PD-1 and PD-L2;
d)能够刺激免疫细胞中IL-2、TNF-α和/或IFN-γ的分泌；d) can stimulate the secretion of IL-2, TNF-α and/or IFN-γ in immune cells;
e)能够抑制肿瘤生长和/或肿瘤细胞增殖；e) capable of inhibiting tumor growth and/or tumor cell proliferation;
f)能够提高免疫细胞的杀伤能力。f) can improve the killing ability of immune cells.
在某些实施方式中，所述的分离的抗原结合蛋白具有下述性质中的一种或多种：In certain embodiments, the isolated antigen binding protein has one or more of the following properties:
a)能够结合人的PD-1；a) capable of binding to human PD-1;
b)能够阻断PD-1和PD-L1结合；b) Can block the binding of PD-1 and PD-L1;
c)能够阻断PD-1和PD-L2结合；c) can block the binding of PD-1 and PD-L2;
d)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；d) can stimulate the secretion of IL-2 and/or IFN-γ in immune cells;
e)能够抑制肿瘤生长和/或肿瘤细胞增殖。e) is capable of inhibiting tumor growth and/or tumor cell proliferation.
另一方面，本申请提供了分离的一种或多种核酸分子，其编码所述的分离的抗原结合蛋白。In another aspect, the application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins.
另一方面，本申请提供了载体，其包含所述的核酸分子。In another aspect, the application provides a vector comprising the nucleic acid molecule.
另一方面，本申请提供了细胞，其包含所述的核酸分子或所述的载体。In another aspect, the application provides a cell comprising the nucleic acid molecule or the vector.
另一方面，本申请提供了制备所述的分离的抗原结合蛋白的方法，所述方法包括在使得所述的分离的抗原结合蛋白表达的条件下，培养所述的细胞。In another aspect, the present application provides a method of preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.
另一方面，本申请提供了药物组合物，其包含所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体和/或所述的细胞，以及任选地药学上可接受的佐剂。In another aspect, the application provides a pharmaceutical composition comprising the isolated antigen binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable adjuvant.
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗PD-1介导的疾病或病症。In another aspect, the present application provides the use of the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in preparing a medicine, the Drugs are used to treat diseases or conditions mediated by PD-1.
在某些实施方式中，所述PD-1介导的疾病或病症包括癌症。In certain embodiments, the PD-1 mediated disease or disorder comprises cancer.
另一方面，本申请提供了抑制PD-1与PD-L1结合的方法，其包括向有需要的受试者施用有效量的所述的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。In another aspect, the present application provides a method for inhibiting the binding of PD-1 to PD-L1, comprising administering an effective amount of the antigen-binding protein, the nucleic acid molecule, and the carrier to a subject in need thereof , the cell and/or the pharmaceutical composition.
另一方面，本申请提供了抑制PD-1与PD-L2结合的方法，其包括向有需要的受试者施用有效量的所述的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。In another aspect, the present application provides a method for inhibiting the binding of PD-1 to PD-L2, comprising administering an effective amount of the antigen-binding protein, the nucleic acid molecule, and the carrier to a subject in need thereof , the cell and/or the pharmaceutical composition.
另一方面，本申请提供了预防、缓解或治疗PD-1介导的疾病或病症的方法，其包括向有需要的受试者施用有效量的所述的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。In another aspect, the present application provides a method of preventing, alleviating or treating a PD-1-mediated disease or disorder, comprising administering to a subject in need thereof an effective amount of the antigen-binding protein, the nucleic acid molecule , the carrier, the cell and/or the pharmaceutical composition.
在某些实施方式中，所述PD-1介导的疾病或病症包括癌症。In certain embodiments, the PD-1 mediated disease or disorder comprises cancer.
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.
附图说明Description of drawings
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:
图1显示的是本申请所述四价人PD-L1胞外段突变体与PD-1-huIgG1 Fc的亲合力(基于BLI法)检测曲线；Figure 1 shows the affinity (based on BLI method) detection curve of the tetravalent human PD-L1 extracellular segment mutant described in the present application and PD-1-huIgG1 Fc;
图2显示的是6H6和pembrolizumab的huIgG1抗体对PD-1和PD-L1结合的抑制曲线；Figure 2 shows the inhibition curves of 6H6 and pembrolizumab huIgG1 antibodies to PD-1 and PD-L1 binding;
图3显示的是6H6和pembrolizumab的huIgG1抗体与PD-1的结合曲线。Figure 3 shows the binding curves of huIgG1 antibodies of 6H6 and pembrolizumab to PD-1.
图4A-4B显示的是在源于供者A和供者B的肿瘤浸润淋巴细胞(TIL)细胞培养基中加入PD-1抗体的CD107a细胞比例结果图。Figures 4A-4B show the results of the proportion of CD107a cells added to the PD-1 antibody in the tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B.
图5A-5F显示的是在源于供者A和供者B的肿瘤浸润淋巴细胞(TIL)细胞培养基中加入PD-1抗体的细胞因子分泌结果图。Figures 5A-5F are graphs showing the results of cytokine secretion by the addition of PD-1 antibody to tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B.
图6A-6B显示的是混合淋巴细胞反应(MLR)实验检测PD-1抗体对于T淋巴细胞的刺激结果图。Figures 6A-6B show the results of the mixed lymphocyte reaction (MLR) assay to detect the stimulation of T lymphocytes by PD-1 antibody.
图7显示的是加入PD-1抗体对TCR-T细胞的杀伤能力增强结果图。Figure 7 shows the results of the enhanced killing ability of TCR-T cells by adding PD-1 antibody.
具体实施方式Detailed ways
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.
术语定义Definition of Terms
在本申请中，术语“PD-1”通常是指程序性细胞死亡1，也称为“程序性死亡1”、In this application, the term "PD-1" generally refers to programmed cell death 1, also known as "programmed cell death 1",
“CD279”、“分化簇279”、“PD1”、“PDCD1”。PD-1通常在T细胞、B细胞、自然杀伤T细胞、活化的单核细胞和树突细胞(DC)上表达，并参与细胞凋亡。PD-1通常包含一个细胞外IgV结构域，跨膜区和胞内结构域。PD-1可结合两种配体，PD-L1和PD-L2。所述“PD-1”包括任何脊椎动物来源的任何天然PD-1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人和食蟹猴)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-1以及由细胞加工所产生的任何形式的PD-1。PD-1可作为跨膜蛋白或作为可溶性蛋白存在。“PD-1”包括完整的PD-1及其片段，还包括PD-1的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-1共同表位的类似物。人PD1的氨基酸序列显示于UniProt(www.uniprot.org)，登录号Q15116。"CD279", "Cluster of differentiation 279", "PD1", "PDCD1". PD-1 is normally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs) and is involved in apoptosis. PD-1 usually contains an extracellular IgV domain, a transmembrane domain and an intracellular domain. PD-1 binds two ligands, PD-L1 and PD-L2. The "PD-1" includes any native PD-1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rat). The term encompasses "full length", unprocessed PD-1 and any form of PD-1 produced by cellular processing. PD-1 can exist as a transmembrane protein or as a soluble protein. "PD-1" includes complete PD-1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-1, and functional variants, isoforms, derivatives, and analogs of PD-1, Epitope analogs. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org), accession number Q15116.
在本申请中，术语“PD-L1”通常是指程序性细胞死亡1配体1，也可称为B7同源物1、B7-H1、分化簇274、(3)274或CD274，其与PD-1结合后下调T细胞活化和细胞因子分泌。“PD-L1”包括任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人和食蟹猴)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。“PD-L1”包括完整的PD-L1及其片段，还包括PD-L1的功能性变 体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-L1共同表位的类似物。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。完整hPD-L1序列可见于GenBank登录号Q9NZQ7下。In this application, the term "PD-L1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which is associated with PD-1 binding downregulates T cell activation and cytokine secretion. "PD-L1" includes any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rats) ). The term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 produced by cellular processing. PD-L1 can exist as a transmembrane protein or as a soluble protein. "PD-L1" includes complete PD-L1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-L1, and functional variants, isoforms, derivatives, and analogs of PD-L1, as well as those having at least one in common with PD-L1 Epitope analogs. The basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.
在本申请中，术语“分离的”或“纯化的”通常是指至少部分从在其天然状态中通常与其结合的其他分子中分离的分子(例如抗体、核酸等)。“分离的或纯化的多肽”基本上没有其他生物分子，如核酸、蛋白质、脂质、碳水化合物、细胞碎片和生长培养基。“分离的或纯化的核酸”至少部分从在其天然状态中通常位于多核苷酸侧翼的核酸分离。因此，在本申请中认为例如由于重组技术，融合它们通常不结合的调节或编码序列的多核苷酸是分离的。即使当存在于例如宿主细胞的染色体中或核酸溶液中，也认为此类分子是分离的。一般地，术语“分离的”和“纯化的”不旨在指此类物质的完全缺失或水、缓冲液或盐的缺失，除非它们以大量干扰分子的实验或治疗用途的量存在。本申请的抗原结合蛋白和编码本申请的抗原结合蛋白的核酸是分离的/纯化的。In this application, the term "isolated" or "purified" generally refers to a molecule (eg, antibody, nucleic acid, etc.) that is at least partially separated from other molecules to which it is ordinarily associated in its natural state. An "isolated or purified polypeptide" is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, cellular debris, and growth media. An "isolated or purified nucleic acid" is at least partially separated from the nucleic acid that normally flanks the polynucleotide in its natural state. Thus, polynucleotides fused to regulatory or coding sequences to which they are not normally associated are considered isolated in this application, eg, due to recombinant techniques. Such molecules are considered isolated even when present, for example, in the chromosome of a host cell or in a nucleic acid solution. Generally, the terms "isolated" and "purified" are not intended to refer to the complete absence of such substances or the absence of water, buffers or salts, unless they are present in quantities that interfere with the experimental or therapeutic use of the molecule. The antigen binding proteins of the present application and nucleic acids encoding the antigen binding proteins of the present application are isolated/purified.
在本申请中，术语“抗原结合蛋白”被以其广义使用并且意指包含与抗原或靶标结合的一部分并且任选地包含允许抗原结合部分采用促进抗原结合蛋白与抗原结合的构型的构架或框架部分的蛋白质。抗原结合蛋白的实例包括人抗体、人源化抗体；嵌合抗体；重组抗体；单链抗体；双功能抗体；三功能抗体；四功能抗体；Fab片段；F(ab’) 2片段；IgD抗体；IgE抗体；IgM抗体；IgG1抗体；IgG2抗体；IgG3抗体；或IgG4抗体以及其片段。抗原结合蛋白可以包括，例如具有移植的CDR或CDR衍生物的替代蛋白构架或人工构架。此类构架包括，但不限于：抗体来源的构架，其包含被引入以便例如使抗原结合蛋白的三维结构稳定的突变；以及完全合成的构架，其包含例如生物相容性聚合物。参见，例如，Korndorfer等，2003，Proteins：Structure，Function，and Bioinformatics，53(1)：121-129(2003)；Roque等，Biotechnol.Prog.20：639-654(2004)。此外，可使用肽抗体模拟物(“PAM”)，也可使用基于抗体模拟物的构架，所述抗体模拟物利用纤连蛋白组分作为构架。 In this application, the term "antigen binding protein" is used in its broadest sense and is meant to comprise a moiety that binds to an antigen or target and optionally comprises a framework that allows the antigen binding moiety to adopt a configuration that facilitates binding of the antigen binding protein to the antigen or framework part of the protein. Examples of antigen binding proteins include human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; trifunctional antibodies; tetrabodies; Fab fragments; F(ab') 2 fragments; ; IgE antibodies; IgM antibodies; IgG1 antibodies; IgG2 antibodies; IgG3 antibodies; or IgG4 antibodies and fragments thereof. Antigen binding proteins can include, for example, alternative protein frameworks or artificial frameworks with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to: antibody-derived frameworks comprising mutations introduced to stabilize, for example, the three-dimensional structure of the antigen binding protein; and fully synthetic frameworks comprising, for example, biocompatible polymers. See, eg, Korndorfer et al, 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al, Biotechnol. Prog. 20: 639-654 (2004). In addition, peptide antibody mimetics ("PAMs") can be used, as can frameworks based on antibody mimetics that utilize fibronectin components as the framework.
在本申请中，术语“抗体”其以最广泛意义使用，且具体涵盖，但不限于，单克隆抗体(包括包含两条轻链和两条重链的全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人类抗体、嵌合抗体、重链抗体和骆驼化单结构域抗体(如，重链可变结构域抗体)。抗体通常具有免疫球蛋白的结构，可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即 IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链，ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。In this application, the term "antibody" is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelized single domain antibodies (eg, heavy chain variable domain antibodies). Antibodies generally have the structure of immunoglobulins, and may comprise proteins of at least two heavy (HC) and two light (LC) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ, δ, γ chains, respectively , α chain, ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain.
在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。天然重链和轻链的可变结构域各自包含四个FR区(HFR1，HFR2，HFR3，HFR4，LFR1，LFR2，LFR3，LFR4)，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)结合。In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region comprises three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of native heavy and light chains each comprise four FR regions (HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4), mostly in a β-sheet configuration, connected by three CDRs, Loop connections are formed, and in some cases part of a beta-sheet structure. The CDRs in each chain are brought together in close proximity by the FR regions and together with the CDRs from the other chain form the antigen-binding site of the antibody. The constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDRs)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDRs一起形成抗体的抗原结合位点，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991)。在本申请中，使用包含了Kabat定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基(参见表1)。In this application, the term "variable" generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, known as complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portion of the variable domain is called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β-sheet configuration, connected by three CDRs, forming loops connecting, and in some cases forming part of, a β-sheet structure. The CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody, the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions , eg involved in antibody-dependent cytotoxicity of antibodies. In the art, the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Starr, Md. (1991). In the present application, amino acid residues in variable domain sequences and full-length antibody sequences were determined using the rules encompassing the Kabat definition (see Table 1).
表1 本申请抗体基于Kabat定义规则的CDR定义Table 1 CDR definition of the antibody of this application based on Kabat definition rules
KabatKabat ResiduesResidues
LCDR1LCDR1 L24-L34L24-L34
LCDR2LCDR2 L50-L56L50-L56
LCDR3LCDR3 L89-L97L89-L97
HCDR1HCDR1 H31-H35H31-H35
HCDR2HCDR2 H50-H66H50-H66
HCDR3HCDR3 H99-H107H99-H107
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位至第bb位的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位至第bb位的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，第24位至第34位的氨基酸序列；H231-H35可以指从抗体重链N端开始，第31位至第35位的氨基酸序列。Wherein, Laa-Lbb can refer to the amino acid sequence from the N-terminus of the antibody light chain, from aa to bb; Haa-Hbb can refer to the amino acid sequence from aa to bb from the N-terminus of the antibody heavy chain sequence. For example, L24-L34 can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from positions 24 to 34; H231-H35 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain, from the 31st to the 35th amino acid sequence.
在本申请中，术语“Fab”指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区，同上)。Fab片段由一条完整的L链与一条重链的可变区和该H链(V H)的第一恒定区(C H1)组成。 In this application, the term "Fab" refers to an antigen-binding fragment of an antibody. Intact antibodies can be digested with papain as described above. Papain digestion of the antibody yields two identical antigen-binding fragments, the "Fab" fragment, and a residual "Fc" fragment (ie, the Fc region, supra). A Fab fragment consists of a complete L chain with the variable region of a heavy chain and the first constant region (C H 1 ) of the H chain (V H ).
在本申请中，术语“Fab′片段”是指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。In this application, the term "Fab' fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment. For example, a Fab' fragment includes all light chains, all heavy chain variable regions, and all or part of the first and second constant regions of the heavy chain. For example, Fab' fragments may also include part or all of the 220-330 amino acid residues of the heavy chain.
在本申请中，术语“F(ab')2”指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。In this application, the term "F(ab')2" refers to antibody fragments produced by pepsin digestion of whole antibodies. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. F(ab')2 fragments have bivalent antigen-binding activity and are capable of cross-linking antigens.
在本申请中，术语“Fv片段”是指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。In the present application, the term "Fv fragment" refers to a monovalent antigen-binding fragment of a human monoclonal antibody, comprising all or part of the heavy and light chain variable regions, and lacking the heavy and light chain constant regions. Heavy chain variable regions and light chain variable regions include, for example, CDRs. For example, Fv fragments include all or part of the amino-terminal variable regions of the heavy and light chains of about 110 amino acids.
在本申请中，术语“scFv”通常是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains can be The variable regions are contiguous (eg, via synthetic linkers such as short flexible polypeptide linkers) and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in this application, a scFv may have the VL and VH variable regions described in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include a VL-linker-VH Or VH-linker-VL can be included.
在本申请中，术语“dAb”通常是指具有VH域、VL域或具有VH域或VL域的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490；以及参考例如WO 06/030220、WO 06/003388和DomantisLtd的其它公布的专利申请。In this application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or a VH domain or a VL domain, see eg Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。In this application, the term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible minor natural mutations. Monoclonal antibodies are usually highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies"), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigenic specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins. In some cases, CDR region residues in a human immunoglobulin (acceptor antibody) can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement. In certain instances, FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues. In addition, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
在本申请中，术语“参比抗体”通常是指任何可以与抗原(例如，PD-1)结合的抗体。在某些情形中，本申请所述抗原结合蛋白可以与参比抗体竞争结合抗原(例如，PD-1)。In this application, the term "reference antibody" generally refers to any antibody that can bind to an antigen (eg, PD-1). In certain instances, the antigen binding proteins described herein can compete with a reference antibody for binding to an antigen (eg, PD-1).
在本申请中，术语“分离的核酸分子”或“分离的多核苷酸”通产是指基因组、mRNA、cDNA或合成来源的DNA或RNA或其一定组合，其不与在自然界中发现的多核苷酸的全部或一部分缔合，或连接至其在自然界中不连接的多核苷酸。In this application, the term "isolated nucleic acid molecule" or "isolated polynucleotide" generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, which is not related to the polynucleus found in nature All or a portion of the nucleotides are associated, or linked, to polynucleotides to which they are not linked in nature.
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a variety of the above-mentioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是293T细胞细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。In this application, the term "cell" generally refers to an individual cell, cell line or cell that can or already contains a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. cell culture. The cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein. The cells can be obtained by transfecting cells in vitro using the vectors described herein. The cells may be prokaryotic cells (eg E. coli) or eukaryotic cells (eg yeast cells, eg COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells). In certain instances, the cells can be mammalian cells. For example, the mammalian cells can be 293T cells. In this application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cells include not only certain specific cells, but also progeny of these cells.
在本申请中，术语“药学上可接受的佐剂”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是pH缓冲水溶液。生理学可接受载体的例子可包括缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，诸如钠；和/或非离子表面活性剂。In this application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are free of the cells or mammals to which they are exposed at the doses and concentrations employed. poisonous. Typically, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, Sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。In the present application, the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，特异性结合PD-1蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白 质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。In the present application, the variant may be a substitution, deletion or addition of one or more in the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the PD-1 protein). A protein or polypeptide of multiple amino acids. For example, the functional variant may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration. For example, the substitutions can be conservative substitutions.
在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，特异性结合PD-1蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。In the present application, the homologue may be at least about 85% of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the PD-1 protein) (eg, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) Proteins or polypeptides with sequence homology.
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待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，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，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。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。In this application, the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences. ) or 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) To obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to yield percent sequence homology. Alignment to determine percent sequence homology can be accomplished 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 homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Protein Similarity Search", Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215: 403-410 , 1990.
在本申请中，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。In this application, the terms "optional" or "optionally" mean that the subsequently described event or circumstance can, but need not, occur.
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。In this application, the term "comprising" generally refers to the meaning of including, encompassing, containing or encompassing. In some cases, it also means "for" and "consisting of".
在本申请中，术语“约”和“大约”应当通常意指鉴于测量的性质或精度，所测量的量的可接受误差程度。示例性误差程度在所给出的值或值范围的20百分数(％)范围内，一般在其10％范围内和更一般在其5％范围内。In this application, the terms "about" and "approximately" shall generally mean an acceptable degree of error in the quantity measured given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%) of a given value or range of values, generally within 10% thereof, and more generally within 5% thereof.
在本申请中，术语“治疗有效量”是指当施用于人或动物时引起足以在所述人或动物中产生治疗效果的应答的抗体的量。有效量易于由本领域普通技术人员按照常规方法确定。In the present application, the term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a human or animal, elicits a response sufficient to produce a therapeutic effect in said human or animal. An effective amount can be readily determined by one of ordinary skill in the art according to routine methods.
发明详述Detailed description of the invention
抗原结合蛋白antigen binding protein
一方面，本申请提供一种抗原结合蛋白，所述抗原结合蛋白可包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:7或9所示的氨基酸序列。In one aspect, the present application provides an antigen-binding protein, which may comprise at least one CDR in the VH of an antibody heavy chain variable region, the VH comprising the amino acid sequence shown in SEQ ID NO: 7 or 9.
本申请所述的抗原结合蛋白包括抗体或其抗原结合片段。本申请所述的抗体可以为单克隆抗体、嵌合抗体、人源化抗体和/或全人源抗体。本申请所述抗体的抗原结合片段可以为Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。 Antigen-binding proteins described herein include antibodies or antigen-binding fragments thereof. The antibodies described herein can be monoclonal antibodies, chimeric antibodies, humanized antibodies and/or fully human antibodies. Antigen-binding fragments of the antibodies described herein may be Fab, Fab', Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAbs.
本申请所述的抗原结合蛋白可以与参比抗体竞争结合PD-1。所述参比抗体可包含轻链可变区和重链可变区。例如，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列；所述LCDR3包含SEQ ID NO:6所示的氨基酸序列；且所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。The antigen binding protein described in this application can compete with the reference antibody for binding to PD-1. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5; The LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6; and the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; the The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1，HCDR2和HCDR3。The antigen binding proteins described herein may comprise the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3.
在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:3所示的氨基酸序列。In the present application, the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3.
在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:2所示的氨基酸序列。In the present application, the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:2.
在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:1所示的氨基酸序列。In the present application, the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1.
在本申请中，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列。In the present application, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.
本申请所述抗原结合蛋白还可包含重链框架区HFR1，HFR2，HFR3和HFR4。The antigen binding proteins described herein may also comprise heavy chain framework regions HFR1, HFR2, HFR3 and HFR4.
在本申请中，所述抗原结合蛋白的HFR1可包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列，且所述HFR1的C末端与所述HCDR1的N末端直接或间接相连。In the present application, the HFR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 19, and the C-terminus of the HFR1 is directly or indirectly linked to the N-terminus of the HCDR1.
在本申请中，所述抗原结合蛋白的HFR2可包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列，且所述HFR2位于所述HCDR1与所述HCDR2之间。In the present application, the HFR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 20, and the HFR2 is located between the HCDR1 and the HCDR2.
在本申请中，所述抗原结合蛋白的HFR3可包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列，且所述HFR3位于所述HCDR2与所述HCDR3之间。In the present application, the HFR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 21, and the HFR3 is located between the HCDR2 and the HCDR3.
在本申请中，所述抗原结合蛋白的HFR4可包含SEQ ID NO:14或SEQ ID NO:22所示的氨基酸序列，且所述HFR4的N末端与所述HCDR3的C末端相连。In the present application, the HFR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 22, and the N-terminus of the HFR4 is linked to the C-terminus of the HCDR3.
在本申请中，所述抗原结合蛋白可包含抗体重链可变区(VH)，且所述VH可包含SEQ ID NO:7或9所示的氨基酸序列。In the present application, the antigen binding protein may comprise an antibody heavy chain variable region (VH), and the VH may comprise the amino acid sequence shown in SEQ ID NO: 7 or 9.
在一些实施方式中，所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列。In some embodiments, HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may comprise the amino acids shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, in order sequence.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列。For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 can respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 in turn.
在一些实施方式中，所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的氨基酸序列。In some embodiments, HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein may comprise the amino acids shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 in sequence, respectively sequence.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的氨基酸序列。For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 can respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 in turn.
本申请所述的抗原结合蛋白可包含重链恒定区。The antigen binding proteins described herein may comprise heavy chain constant regions.
在本申请中，所述重链恒定区可包含人IgG的恒定区。在某些情形中，所述重链恒定区可包括人IgG4恒定区和/或人IgG1的重链恒定区。In the present application, the heavy chain constant region may comprise the constant region of human IgG. In certain instances, the heavy chain constant region can include a human IgG4 constant region and/or a human IgG1 heavy chain constant region.
在一些实施方式中，所述重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。In some embodiments, the heavy chain constant region may comprise the amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。For example, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in sequence, respectively, and the heavy chain of the antigen binding protein is constant A region may comprise the amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列，且所述抗原结合蛋白的重链恒定区可包含SEQ ID NO:27或SEQ  ID NO:28中任一项所示的氨基酸序列。For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 may respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the heavy chain constant region of the antigen-binding protein may comprise The amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的氨基酸序列，且所述抗原结合蛋白的重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。For example, the HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the HFR1, HFR2 of the antigen-binding protein , HFR3 and HFR4 may respectively comprise the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively, and the heavy chain constant region of the antigen-binding protein may comprise The amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28.
本申请中所述抗原结合蛋白可包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:8或10所示的氨基酸序列。The antigen binding protein described in the present application may comprise at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence shown in SEQ ID NO: 8 or 10.
在本申请中，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列。In the present application, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in sequence.
在一些实施方式中，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白可包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:8所示的氨基酸序列。In some embodiments, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in order, and the antigen binding protein Can comprise at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO:8.
在一些实施方式中，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白可包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:16所示的氨基酸序列。In some embodiments, the HCDR1, HCDR2 and HCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, in order, and the antigen binding protein Can comprise at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence set forth in SEQ ID NO:16.
本申请所述抗原结合蛋白可包含轻链互补决定区LCDR1，LCDR2和LCDR3。The antigen binding proteins described herein may comprise light chain complementarity determining regions LCDR1, LCDR2 and LCDR3.
在本申请中，所述抗原结合蛋白的LCDR1包含SEQ ID NO:4所示的氨基酸序列。In the present application, the LCDR1 of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:4.
在本申请中，所述抗原结合蛋白的LCDR2包含SEQ ID NO:5所示的氨基酸序列。In the present application, the LCDR2 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:5.
在本申请中，所述抗原结合蛋白的LCDR3包含SEQ ID NO:6所示的氨基酸序列。In the present application, the LCDR3 of the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:6.
在本申请中，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列。In the present application, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in sequence.
在一些实施方式中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3和LCDR1、LCDR2和LCDR3，其中，所述HCDR1、HCDR2、HCDR3和LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列。In some embodiments, the antigen binding protein may comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 may comprise SEQ ID NO: 1, The amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
在一些实施方式中，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的VH可包含SEQ ID NO:7所示的氨基酸序列。In some embodiments, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein The VH may comprise the amino acid sequence shown in SEQ ID NO:7.
在一些实施方式中，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含 SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的VH可包含SEQ ID NO:9所示的氨基酸序列。In some embodiments, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein The VH may comprise the amino acid sequence shown in SEQ ID NO:9.
在一些实施方式中，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的VH可包含SEQ ID NO:7所示的氨基酸序列，且所述抗原结合蛋白可包含重链恒定区，且所述重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。In some embodiments, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein The VH may comprise the amino acid sequence shown in SEQ ID NO:7, and the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise any of SEQ ID NO:27 or SEQ ID NO:28 An amino acid sequence shown.
在一些实施方式中，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的VH可包含SEQ ID NO:9所示的氨基酸序列，且所述抗原结合蛋白可包含重链恒定区，且所述重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。In some embodiments, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in order, respectively, and the antigen binding protein The VH may comprise the amino acid sequence shown in SEQ ID NO:9, and the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise any of SEQ ID NO:27 or SEQ ID NO:28 An amino acid sequence shown.
本申请所述抗原结合蛋白可包含轻链框架区LFR1，LFR2，LFR3和LFR4。The antigen binding proteins described herein may comprise light chain framework regions LFR1, LFR2, LFR3 and LFR4.
在本申请中，所述抗原结合蛋白的LFR1可包含SEQ ID NO:15或SEQ ID NO:23所示的氨基酸序列，且所述LFR1的C末端与所述LCDR1的N末端直接或间接相连。In the present application, the LFR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 23, and the C-terminus of the LFR1 is directly or indirectly linked to the N-terminus of the LCDR1.
在本申请中，所述抗原结合蛋白的LFR2可包含SEQ ID NO:16或SEQ ID NO:24所示的氨基酸序列，且所述LFR2位于所述LCDR1与所述LCDR2之间。In the present application, the LFR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 24, and the LFR2 is located between the LCDR1 and the LCDR2.
在本申请中，所述抗原结合蛋白的LFR3可包含SEQ ID NO:17或SEQ ID NO:25所示的氨基酸序列，且所述LFR3位于所述LCDR2与所述LCDR3之间。In the present application, the LFR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 25, and the LFR3 is located between the LCDR2 and the LCDR3.
在本申请中，所述抗原结合蛋白的LFR4可包含SEQ ID NO:18或SEQ ID NO:26所示的氨基酸序列，且所述LFR4的N末端与所述LCDR3的C末端相连。In the present application, the LFR4 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 26, and the N-terminus of the LFR4 is linked to the C-terminus of the LCDR3.
在本申请中，所述的抗原结合蛋白可包含VL，且所述VL可包含SEQ ID NO:8或10所示的氨基酸序列。In the present application, the antigen binding protein may comprise VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 8 or 10.
在一些实施方式中，所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。In some embodiments, the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 in sequence .
例如，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。For example, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in sequence, and LFR1, LFR2 of the antigen binding protein , LFR3 and LFR4 in turn may comprise the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序 列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise SEQ ID NO: 15. The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
在一些实施方式中，所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的氨基酸序列。In some embodiments, the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, in sequence .
例如，所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的氨基酸序列。For example, LCDR1, LCDR2 and LCDR3 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in sequence, and LFR1, LFR2 of the antigen binding protein , LFR3 and LFR4 in turn may comprise the amino acid sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的氨基酸序列。For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein can comprise SEQ ID NO: 23. The amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:7或15所示的氨基酸序列，且所述VL可包含SEQ ID NO:8或10所示的氨基酸序列。In the present application, the antigen binding protein may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 7 or 15, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 8 or 10 amino acid sequence.
在一些实施方式中，所述VH可包含SEQ ID NO:7所示的氨基酸序列，且所述VL可包含SEQ ID NO:8所示的氨基酸序列。In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:7, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:8.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列。For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn, and HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: : 11, SEQ ID NO: 12, SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14, and the LFR1, LFR2, LFR3 and LFR4 of the antigen-binding protein may comprise SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: 14 in turn The amino acid sequences shown in ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.
在一些实施方式中，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:10所示的氨基酸序列。In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:9, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:10.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:19、 SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的氨基酸序列。所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的氨基酸序列。For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn, and HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: Amino acid sequences shown in : 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22. The LFR1, LFR2, LFR3 and LFR4 of the antigen binding protein may comprise the amino acid sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 in turn.
在本申请中，所述抗原结合蛋白可包含重链恒定区，且所述重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。In the present application, the antigen binding protein may comprise a heavy chain constant region, and the heavy chain constant region may comprise the amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
在一些实施方式中，所述VH可包含SEQ ID NO:7所示的氨基酸序列，且所述VL可包含SEQ ID NO:8所示的氨基酸序列，且所述抗原结合蛋白的重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:7, the VL can comprise the amino acid sequence set forth in SEQ ID NO:8, and the heavy chain constant region of the antigen binding protein The amino acid sequence set forth in any one of SEQ ID NO:27 or SEQ ID NO:28 may be included.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的氨基酸序列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17和SEQ ID NO:18所示的氨基酸序列，且所述VH可包含SEQ ID NO:7所示的氨基酸序列，且所述VL可包含SEQ ID NO:8所示的氨基酸序列，且所述抗原结合蛋白的重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn, and HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: : 11, SEQ ID NO: 12, SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14, and the LFR1, LFR2, LFR3 and LFR4 of the antigen-binding protein may comprise SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: 14 in turn ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 7, and the VL may comprise SEQ ID NO: 8 The amino acid sequence shown, and the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
在一些实施方式中，所述VH可包含SEQ ID NO:9所示的氨基酸序列，且所述VL可包含SEQ ID NO:10所示的氨基酸序列。In some embodiments, the VH can comprise the amino acid sequence set forth in SEQ ID NO:9, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:10.
例如，所述抗原结合蛋白的HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的氨基酸序列，且所述抗原结合蛋白的LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的氨基酸序列，且所述抗原结合蛋白的HFR1，HFR2，HFR3和HFR4可分别依次包含SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的氨基酸序列，且所述抗原结合蛋白的LFR1，LFR2，LFR3和LFR4依次可包含SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26所示的氨基酸序列，且所述VH可包含SEQ ID NO:15所示的氨基酸序列，且所述VL可包含SEQ ID NO:16所示的氨基酸序列，且所述抗原结合蛋白的重链恒定区可包含SEQ ID NO:27或SEQ ID NO:28中任一项所示的氨基酸序列。For example, HCDR1, HCDR2 and HCDR3 of the antigen-binding protein may respectively comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and LCDR1, LCDR2 of the antigen-binding protein and LCDR3 can respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in turn, and HFR1, HFR2, HFR3 and HFR4 of the antigen binding protein can respectively comprise SEQ ID NO: The amino acid sequences shown in: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, and the LFR1, LFR2, LFR3 and LFR4 of the antigen-binding protein may comprise SEQ ID NO: 23, SEQ ID NO: 23, SEQ ID NO: 22 in turn. ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, and the VH may comprise the amino acid sequence set forth in SEQ ID NO: 15, and the VL may comprise SEQ ID NO: 16 The amino acid sequence shown, and the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:27 or SEQ ID NO:28.
可通过本领域已知的各种测定鉴别、筛选或表征本申请所述的PD-1抗原结合蛋白的物理/化学特性和/或生物活性。The physical/chemical properties and/or biological activities of the PD-1 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.
在一个方面，例如可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白或融合蛋白的抗原结合活性。本申请所述的抗原结合蛋白(例如，PD-1抗体)能够特异性结合PD-1抗原。“特异性结合”PD-1抗原的抗原结合蛋白(例如，PD-1抗体)通常可以结合PD-1，但不结合缺乏PD-1序列的其它蛋白。本申请所述的抗原结合蛋白(例如，PD-1抗体)能够特异性结合PD-1抗原或其标记形式(例如，荧光标记的PD-1抗原)，但不会结合缺乏PD-1表位的其它蛋白。抗原结合蛋白(例如，抗体)是否结合PD-1抗原可使用本领域中已知的任何测定法确定。本领域中已知测定结合亲和力的分析的实例包括生物膜干涉技术(BLI)。In one aspect, the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like Antigen-binding activity of an antigen-binding protein or fusion protein. Antigen binding proteins (eg, PD-1 antibodies) described herein are capable of specifically binding to PD-1 antigen. Antigen-binding proteins (eg, PD-1 antibodies) that "specifically bind" the PD-1 antigen can generally bind PD-1, but not other proteins that lack the PD-1 sequence. Antigen-binding proteins (eg, PD-1 antibodies) described herein are capable of specifically binding PD-1 antigen or a labeled form thereof (eg, fluorescently labeled PD-1 antigen), but not binding epitopes lacking PD-1 of other proteins. Whether an antigen binding protein (eg, an antibody) binds to a PD-1 antigen can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include Biofilm Interferometry (BLI).
本申请所述的抗原结合蛋白可以结合人PD-1蛋白。在某些情形中，本申请所述的抗原结合蛋白还可以与猴(例如，食蟹猴)的PD-1交叉反应。例如，通过流式分析技术和酶联免疫反应所检测的。在本申请中，“交叉反应”是指抗体与来自其它物种的同源蛋白反应的能力。The antigen binding proteins described in this application can bind to human PD-1 protein. In certain instances, the antigen binding proteins described herein can also cross-react with PD-1 of monkeys (eg, cynomolgus monkeys). For example, detected by flow cytometry and ELISA. In this application, "cross-reactivity" refers to the ability of an antibody to react with homologous proteins from other species.
在某些情形中，本申请所述抗原结合蛋白与PD-1的结合活性可使用酶联免疫法测定进行检测。例如，在ELISA中，使用人PD-1抗原蛋白，所述PD-1抗原结合蛋白与PD-1的EC50值可以在约0.0001nM至约100nM之间，例如，可以在约0.001nM至约10nM之间，可以在约0.001nM至约5nM之间，可以在约0.001nM至约1nM之间或可以在约0.01nM至约1nM之间。In some cases, the binding activity of the antigen-binding proteins described herein to PD-1 can be detected using an enzyme-linked immunosorbent assay. For example, in an ELISA using human PD-1 antigen protein, the PD-1 antigen binding protein can have an EC50 value for PD-1 between about 0.0001 nM to about 100 nM, eg, about 0.001 nM to about 10 nM between, can be between about 0.001 nM and about 5 nM, can be between about 0.001 nM and about 1 nM, or can be between about 0.01 nM and about 1 nM.
在另一个方面，本申请所述的抗原结合蛋白能够阻断PD-1与PD-L1的结合。在某些情形中，所述的抗原结合蛋白阻断PD-1与PD-L1的结合酶联免疫法ELISA测定。例如，首先将PD-L1抗原蛋白包被在板上，将递减量的未标记的所述抗原结合蛋白和生物素标记的PD-1蛋白混合后，共同孵育。然后，使用ELISA分析细胞，以证实所述抗原结合蛋白可以阻断PD-1和PD-L1的结合。In another aspect, the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1. In certain instances, the antigen binding protein blocks the binding of PD-1 to PD-L1 in an ELISA assay. For example, first, the PD-L1 antigen protein is coated on the plate, and the unlabeled antigen-binding protein and the biotin-labeled PD-1 protein are mixed with decreasing amounts, and then incubated together. Cells were then analyzed using ELISA to confirm that the antigen binding protein could block the binding of PD-1 and PD-L1.
本申请所述抗原结合蛋白能够刺激免疫细胞中表达CD07a细胞的比例。本申请所述抗原结合蛋白能够刺激免疫细胞中IFN-γ、TNF-α和/或IL2的分泌。例如，本申请所述抗原结合蛋白的刺激能力，可以是在本领域已知激活剂(例如CD3抗体、CD28抗体、或包含CD3抗体和CD28抗体的纳米材料TransAct TM)激活的基础上，增加对免疫细胞激活的效果。所述免疫细胞可包括淋巴细胞，例如B细胞、T细胞，天然杀伤细胞，髓样细胞，例如单核细胞、巨噬细胞、肥大细胞、嗜碱细胞和粒细胞。例如，所述免疫细胞可以包含肿瘤浸润淋巴细胞(TIL)。例如，所述免疫细胞可以包含人工修饰的免疫细胞。例如，所述免疫细胞可以包含TCR-T细胞。可用本领域任何技术人员已知的方法测定免疫细胞中细胞因子的分泌，例如，通过 酶联免疫法(ELISA)定量测定免疫细胞(例如T细胞)增殖情况或由免疫细胞产生的细胞因子(例如由T细胞产生的IFN-γ或IL-2)。例如，可以通过混合淋巴细胞反应(MLR)实验检测PD-1抗体对于T淋巴细胞的刺激结果。 The antigen binding protein described in the present application can stimulate the proportion of CD07a-expressing cells in immune cells. The antigen binding proteins described in the present application can stimulate the secretion of IFN-γ, TNF-α and/or IL2 in immune cells. For example, the stimulating ability of the antigen binding protein described in the present application can be based on the activation of known activators in the art (such as CD3 antibody, CD28 antibody, or nanomaterial TransAct TM comprising CD3 antibody and CD28 antibody), increasing the The effect of immune cell activation. The immune cells may include lymphocytes such as B cells, T cells, natural killer cells, myeloid cells such as monocytes, macrophages, mast cells, basophils and granulocytes. For example, the immune cells may comprise tumor-infiltrating lymphocytes (TILs). For example, the immune cells may comprise artificially modified immune cells. For example, the immune cells may comprise TCR-T cells. The secretion of cytokines from immune cells can be measured by methods known to any person skilled in the art, for example, by enzyme-linked immunosorbent assay (ELISA) to quantify the proliferation of immune cells (eg, T cells) or cytokines produced by immune cells (eg, by enzyme-linked immunosorbent assay). IFN-γ or IL-2 produced by T cells). For example, the stimulation results of PD-1 antibody on T lymphocytes can be detected by mixed lymphocyte reaction (MLR) assay.
核酸分子、载体和细胞Nucleic acid molecules, vectors and cells
另一方面，本申请提供了一种或多种核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。In another aspect, the application provides one or more nucleic acid molecules that can encode the isolated antigen binding proteins described herein. The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。In another aspect, the present application provides a vector, which can comprise the nucleic acid molecule described herein. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host. Such control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. The vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering. For example, the vector is an expression vector.
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。In another aspect, the present application provides a cell, which may comprise the nucleic acid molecule described herein or the vector described herein. In certain embodiments, each or each host cell may comprise one or one nucleic acid molecule or vector described herein. In certain embodiments, each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like. The vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
药物组合物pharmaceutical composition
另一方面，本申请提供了一种药物组合物，其可包含本申请所述的抗原结合蛋白和/或所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的佐剂。所述药学上可接受的佐剂在所采用的剂量和浓度下对接受者无毒性，并且可包括缓冲剂，抗氧化剂，防腐剂，低分子量(小于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，碳水化合物，成盐反离子，金属络合物，和/或非离子表面活性剂。本申请中的药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。In another aspect, the application provides a pharmaceutical composition, which can comprise the antigen binding protein and/or the nucleic acid molecule described herein, the carrier, the host cell, and optionally a pharmaceutical acceptable adjuvants. The pharmaceutically acceptable adjuvants are not toxic to recipients at the doses and concentrations employed, and may include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, pro- Aqueous polymers, amino acids, carbohydrates, salt-forming counterions, metal complexes, and/or nonionic surfactants. The pharmaceutical compositions herein may also contain more than one active compound, typically those active compounds with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述抗原结合蛋白。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。The pharmaceutical compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antigen binding protein. The prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
制备方法Preparation
另一方面，本申请提供了制备所述的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。In another aspect, the present application provides methods for preparing the antigen binding proteins. The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白。例如，可以用连接或天然存在的PD-1蛋白或其片段，免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application. For example, an animal can be immunized with a linked or naturally occurring PD-1 protein or fragment thereof. Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
任何合适形式的PD-1都可以作为免疫原(抗原)，用于产生对PD-1特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人PD-1，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。Any suitable form of PD-1 can be used as an immunogen (antigen) for generating non-human antibodies specific for PD-1 and screening the antibodies for biological activity. The priming immunogen can be full-length mature human PD-1, including native homodimers, or peptides containing single/multiple epitopes. The immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体是IgG抗体，使用IgG1亚型。可以通过用下文实施例中描述的生物学测定筛选抗体实现必需恒定结构域序列的优化，以产生所需生物学活性。同样，任一类轻链都可以在本申请的化合物和方法中使用。具体地说，κ、λ链或其变体在本申请的化合物和方法中是可以用的。Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE. In this application, the antibody is an IgG antibody, and the IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below. Likewise, any type of light chain can be used in the compounds and methods of the present application. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present application.
本申请的抗原结合蛋白或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。The sequences of the DNA molecules of the antigen-binding proteins or fragments thereof of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该核酸分子引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation. The nucleic acid molecule can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
本申请还涉及包含上述的适当核酸分子以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。例如，动物 细胞可以包括(但并不限于)：293T细胞。The present application also relates to vectors comprising suitable nucleic acid molecules as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins. Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. For example, animal cells can include (but are not limited to): 293T cells.
本申请中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本申请的核酸分子所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。通常，在适合本申请抗原结合蛋白表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的抗原结合蛋白。The steps of transforming host cells with recombinant DNA described in this application can be performed using techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultured with conventional media under appropriate conditions. Typically, the transformed host cells are cultured under conditions suitable for expression of the antigen binding proteins of the present application. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. skilled in the art The antigen-binding protein of the present application can be obtained by conventional separation and purification methods well known to those skilled in the art.
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如流式细胞分选技术(FACS)或酶联免疫吸附测定(ELISA))来测定。The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as flow cytometric sorting (FACS) or enzyme-linked immunosorbent assay (ELISA).
方法和用途method and use
另一方面，本申请提供了一种所述抗原结合蛋白在制备药物中的用途。所述抗原结合蛋白可以单独施用或与一种或多种另外的疗法组合施用，另外的疗法例如化疗放射治疗、免疫治疗、手术干预或这些的任何组合。所述药物可用于治疗PD-1介导的疾病或病症，例如，用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。In another aspect, the present application provides a use of the antigen-binding protein in the preparation of a medicine. The antigen binding protein can be administered alone or in combination with one or more additional therapies, such as chemoradiation therapy, immunotherapy, surgical intervention, or any combination of these. The medicament can be used to treat a PD-1 mediated disease or disorder, eg, to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
另一方面，本申请提供的所述抗原结合蛋白，可用于预防或治疗PD-1介导的疾病或病症。所述预防或治疗疾病或病症可以指抑制或延缓疾病或病症的发展或进展。例如，可以用于抑制肿瘤的发展或进展。例如，可以抑制肿瘤增长或肿瘤细胞增殖。On the other hand, the antigen-binding proteins provided in the present application can be used to prevent or treat diseases or conditions mediated by PD-1. The preventing or treating a disease or disorder may refer to inhibiting or delaying the development or progression of the disease or disorder. For example, it can be used to inhibit the development or progression of tumors. For example, tumor growth or tumor cell proliferation can be inhibited.
另一方面，本申请提供了用于预防或治疗PD-1介导的疾病或病症的所述的抗原结合蛋白。In another aspect, the present application provides said antigen binding proteins for use in the prevention or treatment of PD-1 mediated diseases or disorders.
另一方面，本申请提供了抑制PD-1与PD-L1结合的方法，包括施用本申请所述的抗原结合蛋白。所述方法可以是离体或体外方法。在某些情形中，所述方法可包括使生物样品与本申请所述的抗原结合蛋白和/或PD-L1在容许所述抗原结合蛋白和/或PD-1结合PD-L1的条件下接触，检测在所述抗原结合蛋白与PD-1之间是否形成复合物，和检测PD-1与PD-L1之间是否形成复合物。In another aspect, the present application provides a method of inhibiting the binding of PD-1 to PD-L1, comprising administering the antigen binding proteins described herein. The method can be an ex vivo or in vitro method. In certain instances, the method can include contacting a biological sample with an antigen binding protein and/or PD-L1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind PD-L1 , detecting whether a complex is formed between the antigen binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1.
另一方面，本申请提供了预防、缓解或治疗PD-1介导的疾病或病症的方法，其包括向有需要的受试者施用本申请所述的抗体或其抗原结合片段、所述的分子核酸、所述的载体、所述的宿主细胞和/或所述的药物组合物。例如，可以用于抑制肿瘤的发展或进展。例如，可以抑制肿瘤增长或肿瘤细胞增殖。In another aspect, the present application provides a method of preventing, alleviating or treating a PD-1 mediated disease or condition, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof described herein, the Molecular nucleic acid, the vector, the host cell and/or the pharmaceutical composition. For example, it can be used to inhibit the development or progression of tumors. For example, tumor growth or tumor cell proliferation can be inhibited.
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本发明的范围。Without intending to be limited by any theory, the following examples are only used to illustrate the fusion protein, preparation method and use of the present application, and are not used to limit the scope of the present invention.
实施例Example
实施例1:重组人程序性死亡受体1(PD-1)25位至167位氨基酸序列与人IgG1 Fc区的融合蛋白(PD-1-huIgG1 Fc)的表达载体构建与真核表达Example 1: Expression vector construction and eukaryotic expression of the fusion protein (PD-1-huIgG1 Fc) of recombinant human programmed death receptor 1 (PD-1) amino acid sequence 25 to 167 and human IgG1 Fc region
1.PD-1 25位至167位氨基酸区间的基因序列的合成及PD-1-huIgG1 Fc融合蛋白的表达载体的构建1. Synthesis of the gene sequence of the amino acid range from 25 to 167 of PD-1 and construction of the expression vector of PD-1-huIgG1 Fc fusion protein
通过化学合成的方式合成程序性死亡受体1(PD-1)(NCBI accession No.NP_005009.2)的第25位异亮氨酸至第167位谷氨酰胺区间的基因序列(SEQ ID NO:29)，该基因序列编码SEQ ID NO:30所示的氨基酸序列。通过化学合成的方式合成人IgG1重链恒定区的第100位脯氨酸至第330位赖氨酸氨酸区间的基因序列。化学合成含小鼠Igkv3-10信号肽基因序列的上游引物用于表达载体构建。通过分子克隆，将PD-1基因片段与人IgG1 Fc基因片段进行拼接。拼接产物用TaKaRa无缝克隆试剂盒克隆到pCDNA3.1(Thermo)中。The gene sequence from the 25th isoleucine to the 167th glutamine of the programmed death receptor 1 (PD-1) (NCBI accession No.NP_005009.2) was synthesized by chemical synthesis (SEQ ID NO: 29), the gene sequence encodes the amino acid sequence shown in SEQ ID NO: 30. The gene sequence from the 100th proline to the 330th lysine in the constant region of the human IgG1 heavy chain was synthesized by chemical synthesis. Chemically synthesized upstream primers containing mouse Igkv3-10 signal peptide gene sequence were used for expression vector construction. Through molecular cloning, the PD-1 gene fragment was spliced with the human IgG1 Fc gene fragment. The spliced product was cloned into pCDNA3.1 (Thermo) using the TaKaRa seamless cloning kit.
2.重组PD-1-huIgG1 Fc融合蛋白的表达与纯化2. Expression and purification of recombinant PD-1-huIgG1 Fc fusion protein
以此表达载体转染293T细胞(ATCC)5天后，收集培养上清，用AKTA explorer 100(GE)纯化重组PD-1-huIgG1 Fc融合蛋白。由于糖基化修饰等原因，重组PD-1-huIgG1 Fc融合蛋白经还原SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约60k道尔顿。After transfecting 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected, and the recombinant PD-1-huIgG1 Fc fusion protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant PD-1-huIgG1 Fc fusion protein was shown to be about 60k Daltons in size by Coomassie brilliant blue staining after reducing SDS-PAGE electrophoresis.
实施例2:重组人程序性死亡受体1(PD-1)25位至167位氨基酸序列与多聚组氨酸标签融合蛋白(PD-1-his)的表达载体构建与真核表达Example 2: Construction of expression vector and eukaryotic expression of recombinant human programmed death receptor 1 (PD-1) amino acid sequence 25 to 167 and polyhistidine tag fusion protein (PD-1-his)
1.PD-1 25位至167位氨基酸区间的基因序列的合成及多聚组氨酸标签融合蛋白的表达载体的构建1. Synthesis of the gene sequence of the amino acid range from 25 to 167 of PD-1 and construction of the expression vector of the polyhistidine tag fusion protein
通过化学合成的方式合成程序性死亡受体1(PD-1)(NCBI accession No.NP_005009.2)的第25位异亮氨酸至第167位谷氨酰胺区间的基因序列。化学合成含小鼠Igkv3-10信号肽基因序列的上游引物和含多聚组氨酸标签(具有SEQ ID NO:32所示的氨基酸序列)基因序列(SEQ ID NO:31)的下游引物用于构建表达PD-1-his的pCDNA3.1载体。The gene sequence from the 25th isoleucine to the 167th glutamine of programmed death receptor 1 (PD-1) (NCBI accession No. NP_005009.2) was synthesized by chemical synthesis. The upstream primer containing the mouse Igkv3-10 signal peptide gene sequence and the downstream primer containing the polyhistidine tag (having the amino acid sequence shown in SEQ ID NO:32) gene sequence (SEQ ID NO:31) were chemically synthesized for The pCDNA3.1 vector expressing PD-1-his was constructed.
2.重组PD-1-his蛋白的表达与纯化2. Expression and purification of recombinant PD-1-his protein
以此表达载体转染293T细胞(ATCC)5天后，收集培养上清，用AKTA explorer 100(GE)纯化重组PD-1-his蛋白。由于糖基化修饰等原因，重组PD-1-his蛋白经还原SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约40k道尔顿。After transfecting 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected, and the recombinant PD-1-his protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant PD-1-his protein was shown to be about 40k Daltons in size by Coomassie brilliant blue staining after reducing SDS-PAGE electrophoresis.
实施例3：抗人PD-1抗体(Nivolumab)表达载体构建与真核表达Example 3: Construction of anti-human PD-1 antibody (Nivolumab) expression vector and eukaryotic expression
1.Nivolumab抗体可变区基因的获取及表达载体构建1. Acquisition of variable region gene of Nivolumab antibody and construction of expression vector
通过化学合成的方式合成Nivolumab抗体轻重链可变区基因分别为SEQ ID NO:33和 SEQ ID NO:34，Nivolumab抗体轻链可变区具有SEQ ID NO:35所示的氨基酸序列，Nivolumab抗体重链可变区具有SEQ ID NO:36所示的氨基酸序列。以重链可变区基因为模板，PCR扩增重链可变区片段，扩增产物用TaKaRa无缝克隆试剂盒克隆到含人IL-2信号肽基因和人IgG1重链恒定区基因的pFUSEss-CHIg-hG1(invivogen)中。以轻链可变区基因为模板，PCR扩增轻链可变区片段，扩增产物用TaKaRa无缝克隆试剂盒克隆到含人IL-2信号肽基因和人卡帕轻链恒定区基因的pFUSE2ss-CLIg-hK(invivogen)中。The light and heavy chain variable region genes of Nivolumab antibody were synthesized by chemical synthesis as SEQ ID NO: 33 and SEQ ID NO: 34, respectively. The light chain variable region of Nivolumab antibody has the amino acid sequence shown in SEQ ID NO: 35. Nivolumab antibody heavy The chain variable region has the amino acid sequence shown in SEQ ID NO:36. Using the heavy chain variable region gene as a template, the heavy chain variable region fragment was amplified by PCR, and the amplified product was cloned into pFUSEss containing human IL-2 signal peptide gene and human IgG1 heavy chain constant region gene using TaKaRa seamless cloning kit. -CHIg-hG1 (invivogen). Using the light chain variable region gene as a template, the light chain variable region fragment was amplified by PCR, and the amplified product was cloned into a human IL-2 signal peptide gene and human kappa light chain constant region gene using the TaKaRa seamless cloning kit. pFUSE2ss-CLIg-hK (invivogen).
2.Nivolumab抗体的表达与纯化2. Expression and purification of Nivolumab antibody
以此双质粒1:1比例共转染293T细胞(ATCC)5天后，收集培养上清，用AKTA explorer 100(GE)纯化Nivolumab抗体。Nivolumab抗体经非还原SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约150k道尔顿。After co-transfecting 293T cells (ATCC) with this dual plasmid at a ratio of 1:1 for 5 days, the culture supernatant was collected, and the Nivolumab antibody was purified with AKTA explorer 100 (GE). The Nivolumab antibody was shown to be approximately 150 k Daltons in size by Coomassie brilliant blue staining after non-reducing SDS-PAGE electrophoresis.
实施例4：ELISA检测重组人PD-1(PD-1-huIgG1 Fc或PD-1-his)与Nivolumab抗体结合Example 4: ELISA detection of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) binding to Nivolumab antibody
利用酶联免疫吸附测定(ELISA)来检测重组人PD-1(PD-1-huIgG1 Fc或PD-1-his)与Nivolumab抗体结合，ELISA实验具体操作如下：微孔板中加入上文制备的PD-1-huIgG1 Fc或PD-1-his融合蛋白100ng/孔，4℃包被过夜。PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。100ul PBS洗板后加入100ng/孔Nivolumab嵌合抗体，37℃结合1小时。PBST清洗三遍，加入100ul 1:5000稀释的HRP-山羊抗人IgG(Fab特异性的)37℃结合1小时。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值。OD450数值可反映Nivolumab抗体与重组人PD-1的结合情况。Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of recombinant human PD-1 (PD-1-huIgG1 Fc or PD-1-his) to Nivolumab antibody. PD-1-huIgG1 Fc or PD-1-his fusion protein 100ng/well, coated overnight at 4°C. PBS was washed three times, 1% BSA/PBS was added, 200uL/well, and the cells were blocked at 37°C for 1 hour. After washing the plate with 100ul PBS, add 100ng/well of Nivolumab chimeric antibody, and bind at 37°C for 1 hour. After washing three times with PBST, 100ul of 1:5000 diluted HRP-goat anti-human IgG (Fab-specific) was added for binding at 37°C for 1 hour. Wash three times with PBST, add 100uL/well of TMB chromogenic solution, develop color at 37°C for 10 minutes, add 100uL/well of ELISA stop solution, and read the OD450 value with a microplate reader. The OD450 value can reflect the binding of Nivolumab antibody to recombinant human PD-1.
实施例5：四价人细胞程序性死亡-配体1(PD-L1)胞外段突变体的表达及与人PD-1分子的亲合力测定Example 5: Expression of tetravalent human programmed cell death-ligand 1 (PD-L1) extracellular segment mutants and determination of their affinity with human PD-1 molecules
1.四价人PD-L1胞外段突变体的表达载体的构建1. Construction of expression vector for quadrivalent human PD-L1 extracellular segment mutants
通过化学合成的方式合成由一段柔性肽连接的两个人PD-L1胞外段突变体的基因序列(SEQ ID NO:37)，该基因序列编码SEQ ID NO:38所示的氨基酸序列。通过化学合成的方式合成人IgG1重链恒定区(UniProtKB/Swiss-Prot accession No.P01857.1)的第100位脯氨酸至第330位赖氨酸氨酸区间的基因序列。化学合成含小鼠Igkv3-10信号肽基因序列的上游引物用于表达载体构建。通过分子克隆，将PD-L1胞外段突变体重复结构的基因片段与人IgG1 Fc基因片段进行拼接。拼接产物用TaKaRa无缝克隆试剂盒克隆到pCDNA3.1(Thermo)中。The gene sequence (SEQ ID NO:37) of two human PD-L1 extracellular segment mutants linked by a flexible peptide was synthesized by chemical synthesis, and the gene sequence encoded the amino acid sequence shown in SEQ ID NO:38. The gene sequence from the 100th proline to the 330th lysine of the human IgG1 heavy chain constant region (UniProtKB/Swiss-Prot accession No. P01857.1) was synthesized by chemical synthesis. Chemically synthesized upstream primers containing mouse Igkv3-10 signal peptide gene sequence were used for expression vector construction. Through molecular cloning, the gene fragment of the PD-L1 extracellular segment mutant repeat structure was spliced with the human IgG1 Fc gene fragment. The spliced product was cloned into pCDNA3.1 (Thermo) using the TaKaRa seamless cloning kit.
2.四价人PD-L1胞外段突变体的表达与纯化2. Expression and purification of tetravalent human PD-L1 extracellular segment mutants
以此表达载体转染293T细胞(ATCC)5天后，收集培养上清，用AKTA explorer 100(GE)纯化四价人PD-L1胞外段突变体蛋白。由于糖基化修饰等原因，四价人PD-L1胞外段突变体蛋白经还原SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约85k道尔顿。After transfecting 293T cells (ATCC) with this expression vector for 5 days, the culture supernatant was collected, and the tetravalent human PD-L1 extracellular segment mutant protein was purified with AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the tetravalent human PD-L1 extracellular segment mutant protein was shown to be about 85k Daltons in size by Coomassie brilliant blue staining after reducing SDS-PAGE electrophoresis.
3.PD-1-huIgG1 Fc融合蛋白的生物素化3. Biotinylation of PD-1-huIgG1 Fc fusion protein
应用EZ-Link Sulfo-NHS-LC-Biotin(Thermo)提供的标准操作程序对PD-1-huIgG1 Fc融合蛋白进行随机生物素化。用ELISA方法验证生物素化的PD-1-huIgG1 Fc融合蛋白与Nivolumab抗体的结合活性。PD-1-huIgG1 Fc fusion protein was randomly biotinylated using standard operating procedures provided by EZ-Link Sulfo-NHS-LC-Biotin (Thermo). The binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA.
4.测定四价人PD-L1胞外段突变体与PD-1-huIgG1 Fc的亲合力(avidity)4. Determination of the avidity of tetravalent human PD-L1 extracellular segment mutants with PD-1-huIgG1 Fc
利用Octet K2(ForteBio)分子互作分析仪对四价人PD-L1胞外段突变体与PD-1-huIgG1 Fc的亲合力进行测定。依照Octet K2的标准操作流程，配置100nM的生物素化的PD-1-huIgG1 Fc融合蛋白并将其固化在SA探针(ForteBio)上，固化高度为1nM。以从50nM浓度起始两倍梯度稀释的四价人PD-L1胞外段突变体作为分析物进行实验，测得四价人PD-L1胞外段突变体与PD-1-huIgG1 Fc的亲合力为9.4nM，亲合力测定结果显示于图1。已知野生型PD-1和PD-L1结合亲和力8.2uM(见Cheng,X.et al.,J.Biol.Chem.,288(17):11771-85,2013)。The affinity of tetravalent human PD-L1 extracellular segment mutants to PD-1-huIgG1 Fc was determined using Octet K2 (ForteBio) molecular interaction analyzer. Following the standard operating procedure of Octet K2, 100 nM of biotinylated PD-1-huIgG1 Fc fusion protein was prepared and immobilized on SA probe (ForteBio) at a height of 1 nM. The tetravalent human PD-L1 ecto-segment mutant was used as the analyte in a two-fold serial dilution starting from a concentration of 50 nM, and the affinity of the quadrivalent human PD-L1 ecto-segment mutant with PD-1-huIgG1 Fc was determined. The resultant force was 9.4 nM and the results of the affinity assay are shown in FIG. 1 . Wild-type PD-1 and PD-L1 have a known binding affinity of 8.2 uM (see Cheng, X. et al., J. Biol. Chem., 288(17):11771-85, 2013).
实施例6：抗人PD-1鼠源抗体的制备、鼠抗人源化Example 6: Preparation of anti-human PD-1 mouse antibody, mouse anti-humanization
1.免疫动物1. Immune animals
将2mg/mL的PD-1-huIgG1 Fc融合蛋白作为抗原与等体积的完全弗氏佐剂(Sigma-Aldrich)混合乳化，取10只6周大雌性Balb/c小鼠(进行皮下免疫，每只100ug抗原。在初次免疫后，每十天时间进行一次加强免疫，共执行四次皮下免疫，第五次免疫时直接用PD-1-huIgG1 Fc融合蛋白进行脾脏冲击免疫。2 mg/mL of PD-1-huIgG1 Fc fusion protein as antigen was mixed and emulsified with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), and 10 6-week-old female Balb/c mice (for subcutaneous immunization, each Only 100ug antigen. After the initial immunization, booster immunization was performed every ten days, and a total of four subcutaneous immunizations were performed, and the spleen pulse immunization was directly performed with PD-1-huIgG1 Fc fusion protein in the fifth immunization.
2.血清效价检测2. Serum titer detection
每次加强免疫前尾静脉取血50uL，离心去除细胞，保留血清。ELISA微孔板中加入PD-1-his(ACRO Biosystems)50ng/孔，4℃包被过夜。PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。加入梯度稀释的小鼠血清，37℃结合1小时。PBST清洗三遍，加入100ul 1:5000稀释的HRP-山羊抗小鼠IgG(上海翊胜)37℃结合1小时。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值。Before each booster immunization, 50uL of blood was collected from the tail vein, and the cells were removed by centrifugation, and the serum was retained. 50ng/well of PD-1-his (ACRO Biosystems) was added to the ELISA microplate and coated overnight at 4°C. PBS was washed three times, 1% BSA/PBS was added, 200uL/well, and the cells were blocked at 37°C for 1 hour. Serially diluted mouse serum was added and combined for 1 hour at 37°C. After washing three times with PBST, 100ul of HRP-goat anti-mouse IgG (Shanghai Yisheng) diluted at 1:5000 was added for binding at 37°C for 1 hour. Wash three times with PBST, add 100uL/well of TMB chromogenic solution, develop color at 37°C for 10 minutes, add 100uL/well of ELISA stop solution, and read the OD450 value with a microplate reader.
3.构建免疫文库3. Construction of immune library
3.1小鼠脾细胞总cDNA获取3.1 Obtaining total cDNA of mouse splenocytes
在用PD-1-huIgG1 Fc融合蛋白直接进行腹腔注射方式进行冲击免疫后四天处死小鼠，取 脾脏。用70微米细胞筛网(BD)研磨整个脾脏，获取脾脏细胞。用PBS冲洗两遍后，1000g离心10分钟，获取脾脏细胞。使用Trizol RNA提取试剂盒抽提总RNA。The mice were sacrificed four days after the direct intraperitoneal injection with PD-1-huIgG1 Fc fusion protein for shock immunization, and the spleen was removed. Spleen cells were obtained by triturating the whole spleen with a 70 micron cell mesh (BD). After washing twice with PBS, spleen cells were obtained by centrifugation at 1000g for 10 minutes. Total RNA was extracted using Trizol RNA extraction kit.
以所述RNA为模板，使用SuperScript TM IV First-Strand Synthesis System试剂盒合成第一链cDNA。 Using the RNA as a template, the first-strand cDNA was synthesized using the SuperScript ™ IV First-Strand Synthesis System kit.
3.2抗体基因扩增与轻重链拼接3.2 Antibody gene amplification and light and heavy chain splicing
以所述cDNA为模板，参考文献所述的抗体扩增引物(Schaefer J.V.,Honegger A.,Plückthun A.(2010)Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.In:Kontermann R.,Dübel S.(eds)Antibody Engineering.Springer Protocols Handbooks.Springer,Berlin,Heidelberg)，使用重链可变域上游引物和下游引物PCR扩增重链可变域基因，使用轻链可变域上游引物和下游引物引物PCR扩增卡帕链可变域基因。在50uL反应体系中，分别加入25uL phusion master mix(Thermo)，上游引物2.5uL(25pmol)，下游引物2.5uL(25pmol)，1.5uL DMSO，0.5uL cDNA和18uL ddH2O。按以下程序进行PCR反应：98℃预变性1分钟后进入温度循环，98℃变性30秒，58℃退火30秒，72℃延伸1分钟，循环30次，72℃最终延伸10分钟。Taking the cDNA as a template, the antibody amplification primers described in the reference (Schaefer J.V., Honegger A., Plückthun A. (2010) Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.In: Kontermann R., Dübel S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg), use heavy chain variable domain upstream primers and downstream primers to PCR amplify heavy chain variable domain genes, use light chain variable domain upstream primers and downstream primers Primer primers PCR amplified the kappa chain variable domain gene. In the 50uL reaction system, 25uL phusion master mix (Thermo), 2.5uL (25pmol) upstream primer, 2.5uL (25pmol) downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O were added respectively. The PCR reaction was performed as follows: pre-denaturation at 98°C for 1 minute followed by temperature cycling, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
使用DNA胶回收试剂盒回收扩增得到的VH基因和VL基因。将等量的VH基因和VL基因混合后为模板，利用上游引物scFv-F和下游引物scFv-R通过重叠PCR扩增scFv基因。在50uL反应体系中，分别加入25uL phusion master mix，上游引物2.5uL(25pmol)，下游引物2.5uL(25pmol)，1.5uL DMSO，0.5uL cDNA和18uL ddH2O。按以下程序进行PCR反应：98℃预变性1分钟后进入温度循环，98℃变性30秒，58℃退火30秒，72℃延伸1分钟，循环30次，72℃最终延伸10分钟。The amplified VH and VL genes were recovered using a DNA gel recovery kit. The scFv gene was amplified by overlapping PCR using the upstream primer scFv-F and the downstream primer scFv-R after mixing the same amount of VH gene and VL gene as a template. In the 50uL reaction system, add 25uL phusion master mix, 2.5uL (25pmol) upstream primer, 2.5uL (25pmol) downstream primer, 1.5uL DMSO, 0.5uL cDNA and 18uL ddH2O. The PCR reaction was performed as follows: pre-denaturation at 98°C for 1 minute followed by temperature cycling, denaturation at 98°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
使用DNA胶回收试剂盒回收扩增得到的scFv基因片段。The amplified scFv gene fragments were recovered using a DNA gel recovery kit.
3.3构建免疫文库3.3 Construction of immune library
分别使用SfiI DNA内切酶消化scFv基因片段和pcomb3XTT载体(美国Scripps研究所)。在50uL反应体系中，分别加入SfiI 2uL，10x缓冲液5uL，DNA 3ug，加ddH2O至50uL。充分混匀后，50℃孵育3小时。The scFv gene fragment and pcomb3XTT vector (Scripps Research Institute, USA) were digested with SfiI endoDNA, respectively. In the 50uL reaction system, add SfiI 2uL, 10x buffer 5uL, DNA 3ug, and add ddH2O to 50uL. After mixing well, incubate at 50°C for 3 hours.
使用DNA胶回收试剂盒回收酶切后的scFv基因片段和pcomb3XTT载体。使用T4连接酶环化酶切后的scFv基因片段和酶切后的pcomb3XTT载体。在50uL反应体系中，分别加入T4连接酶1uL，10x缓冲液5uL，scFv基因100ng，pComb3XTT载体500ng，加ddH2O至50uL。充分混匀后，4℃孵育16小时。取少量产物通过琼脂糖凝胶电泳验证连接效率。The digested scFv gene fragment and pcomb3XTT vector were recovered using DNA gel recovery kit. The scFv gene fragment and the digested pcomb3XTT vector were digested with T4 ligase cyclase. In a 50uL reaction system, add 1uL of T4 ligase, 5uL of 10x buffer, 100ng of scFv gene, 500ng of pComb3XTT vector, and add ddH2O to 50uL. After thorough mixing, incubate at 4°C for 16 hours. Take a small amount of product to verify the ligation efficiency by agarose gel electrophoresis.
将10uL上述连接环化产物加入自制的TG1电转化感受态中，然后通过电转仪进行电击 转化。取出10ul电转化后的细菌通过合理的稀释并在含有氨苄青霉素的平板上划线，以此计数并统计噬菌体抗体文库的大小。剩余的电转化后的细菌加入含100ug/mL氨苄青霉素和2％葡萄糖的2xYT培养基，置于加热培养箱培养。培养结束后在4℃以4000G离心10分钟，在沉淀菌中补充适量甘油储存于-80℃作为抗体菌种库。通过多次电转化积累获得超过3E9库容的scFv免疫文库。10uL of the above-mentioned ligation and cyclization product was added to the self-made TG1 electrotransformation competent, and then electroporated by electroporator. Take out 10ul of electrotransformed bacteria and streak them on a plate containing ampicillin by reasonable dilution to count and count the size of the phage antibody library. The remaining electrotransformed bacteria were added to 2xYT medium containing 100ug/mL ampicillin and 2% glucose, and placed in a heated incubator for cultivation. After culturing, centrifuge at 4000G for 10 minutes at 4°C, supplement appropriate amount of glycerol in the precipitated bacteria and store at -80°C as an antibody strain library. The scFv immune library exceeding the 3E9 repertoire was obtained by multiple electroporation accumulation.
4.鼠源免疫抗体噬菌体文库的筛选与鉴定4. Screening and identification of murine immune antibody phage library
4.1 PD-1-huIgG1 Fc融合蛋白的生物素化4.1 Biotinylation of PD-1-huIgG1 Fc fusion protein
应用EZ-Link Sulfo-NHS-LC-Biotin提供的标准操作程序对PD-1-huIgG1 Fc融合蛋白进行随机生物素化。用ELISA方法验证生物素化的PD-1-huIgG1 Fc融合蛋白与Nivolumab抗体的结合活性。PD-1-huIgG1 Fc fusion protein was randomly biotinylated using standard operating procedures provided by EZ-Link Sulfo-NHS-LC-Biotin. The binding activity of biotinylated PD-1-huIgG1 Fc fusion protein to Nivolumab antibody was verified by ELISA.
4.2生物淘选4.2 Biopanning
以PD-1-huIgG1 Fc融合蛋白为目标蛋白应用生物淘选，对上述鼠源免疫抗体文库进行生物淘选获得与PD-1-huIgG1 Fc融合蛋白(尤其是PD-1胞外域)结合的抗体。从抗体菌种库中取100OD细菌以起始OD600＝0.1密度复苏并生长至对数期后应用M13KO7辅助噬菌体挽救抗体文库，离心后用含有氨苄青霉素和卡那霉素的2xYT培养基重悬并在30℃过夜扩增。PEG/NaCl沉淀噬菌体，用甘油/PBST溶解噬菌体沉淀获得免疫库噬菌体悬液。酪蛋白封闭的噬菌体投入酪蛋白封闭的生物素化的huIgG1 Fc(ACRO Biosystems)融合蛋白和酪蛋白封闭的Dynabeads M-270链霉亲合素共孵育体系中，收集上清噬菌体悬液。进一步，将收集到的噬菌体悬液投入酪蛋白封闭的生物素化的PD-1-huIgG1 Fc融合蛋白和酪蛋白封闭的Dynabeads M-270链霉亲合素共孵育体系中，用PBST清洗磁珠去除无法与PD-1-huIgG1 Fc融合蛋白结合的噬菌体。在适当的洗脱条件下，应用自制四价PD-L1胞外段突变体对噬菌体进行竞争性洗脱。留取10ul洗脱的噬菌体溶液用于测定输出噬菌体总量，剩余噬菌体溶液用于感染对数增长的TG1，过夜扩增后视为下一轮淘选使用的抗体文库。生物淘选共执行三轮，淘选实验参数详见表2。Using the PD-1-huIgG1 Fc fusion protein as the target protein, biopanning was performed, and the above-mentioned murine immune antibody library was biopanned to obtain antibodies that bind to the PD-1-huIgG1 Fc fusion protein (especially the extracellular domain of PD-1). . 100 OD bacteria were recovered from the antibody strain library at an initial density of OD600=0.1 and grown to log phase. The antibody library was rescued by M13KO7 helper phage, and resuspended in 2xYT medium containing ampicillin and kanamycin after centrifugation. Amplify overnight at 30°C. The phage was precipitated with PEG/NaCl, and the phage pellet was dissolved with glycerol/PBST to obtain a phage suspension of the immune library. The casein-blocked phage was put into the co-incubation system of casein-blocked biotinylated huIgG1 Fc (ACRO Biosystems) fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the supernatant phage suspension was collected. Further, the collected phage suspension was put into the co-incubation system of casein-blocked biotinylated PD-1-huIgG1 Fc fusion protein and casein-blocked Dynabeads M-270 streptavidin, and the magnetic beads were washed with PBST. Phages that cannot bind to the PD-1-huIgG1 Fc fusion protein are removed. Under appropriate elution conditions, the phage was subjected to competitive elution using a homemade tetravalent PD-L1 ecto-segment mutant. 10ul of the eluted phage solution was used to determine the total amount of output phage, and the remaining phage solution was used to infect the logarithmically growing TG1, which was regarded as the antibody library used in the next round of panning after overnight amplification. A total of three rounds of biopanning were performed, and the parameters of the panning experiments are shown in Table 2.
表2 抗体文库生物淘选实验参数Table 2 Antibody library biopanning experimental parameters
Figure PCTCN2021121785-appb-000001
Figure PCTCN2021121785-appb-000001
Figure PCTCN2021121785-appb-000002
Figure PCTCN2021121785-appb-000002
4.3初筛PD-1胞外域特异性结合且具PD-1-四价PD-L1阻断作用的克隆4.3 Primary screening of clones that specifically bind to the extracellular domain of PD-1 and block PD-1-tetravalent PD-L1
将第三轮生物淘选结束后获得的抗体文库进行稀释涂布于含有氨苄青霉素的平板上获得单克隆，挑选单克隆在深孔板中过夜培养。次日将深孔板进行反复三次冻融，离心上清用于后续两类ELISA反应。The antibody library obtained after the third round of biopanning was diluted and spread on a plate containing ampicillin to obtain monoclones, and the monoclones were selected and cultured overnight in deep-well plates. The next day, the deep-well plate was freeze-thawed three times, and the supernatant was centrifuged for the subsequent two types of ELISA reactions.
检测结合活性的ELISA反应：利用50ng PD-1-his过夜包被，PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。PBS清洗三遍后加入100ul离心上清37℃孵育1小时，PBST清洗三遍后加入100ul 1:5000稀释的HRP偶联的山羊抗鼠IgG(Fab特异性的)(Thermo)。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值。该筛选步骤重复两次独立实验以保证数据准确，选取OD450数均值大于0.15的克隆进入后续分析。ELISA reaction to detect binding activity: coat with 50ng PD-1-his overnight, wash with PBS three times, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, 100ul of the centrifuged supernatant was added and incubated at 37°C for 1 hour. After washing three times with PBST, 100ul of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added at a 1:5000 dilution. Wash three times with PBST, add 100uL/well of TMB chromogenic solution, develop color at 37°C for 10 minutes, add 100uL/well of ELISA stop solution, and read the OD450 value with a microplate reader. This screening step was repeated two independent experiments to ensure accurate data, and clones with an average OD450 value greater than 0.15 were selected for subsequent analysis.
检测阻断活性的ELISA反应：利用PD-1-his过夜包被，PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。PBS清洗三遍后加入100ul离心上清和20ng四价PD-L1混合液37℃孵育1小时，PBST清洗三遍后加入100ul 1:5000稀释的HRP偶联的山羊抗鼠IgG(Fab特异性的)(Thermo)。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值。该筛选步骤重复两次独立实验以保证数据准确，选取OD450数均值小于1的克隆进入后续分析。ELISA reaction for detecting blocking activity: coat with PD-1-his overnight, wash with PBS three times, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, add 100ul of centrifugal supernatant and 20ng of tetravalent PD-L1 mixture and incubate at 37°C for 1 hour. After washing three times with PBST, add 100ul of 1:5000 diluted HRP-conjugated goat anti-mouse IgG (Fab specific) (Thermo). Wash three times with PBST, add 100uL/well of TMB chromogenic solution, develop color at 37°C for 10 minutes, add 100uL/well of ELISA stop solution, and read the OD450 value with a microplate reader. This screening step was repeated two independent experiments to ensure accurate data, and clones with an average OD450 number less than 1 were selected for subsequent analysis.
进一步，将同时符合以上两个条件的候选克隆进行测序，获取抗体轻链可变域和重链可变域序列。Further, the candidate clones that meet the above two conditions at the same time are sequenced to obtain the antibody light chain variable domain and heavy chain variable domain sequences.
4.4复筛PD-1胞外域特异性结合且具PD-1-四价PD-L1阻断作用的克隆4.4 Rescreening of clones that specifically bind to the extracellular domain of PD-1 and block PD-1-tetravalent PD-L1
将初筛获得的候选克隆用50ml摇菌管(thermo)扩大培养，利用溶菌酶(上海生工)结合三次冻融的方法获取周质腔提取物，并再次用两种ELISA鉴定克隆的结合和阻断活性。利用50ng PD-1-his过夜包被，PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。PBS清洗三遍后加入100ul离心上清37℃孵育1小时，PBST清洗三遍后加入100ul 1:5000稀释的HRP偶联的山羊抗鼠IgG(Fab特异性的)(Thermo)。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值，以此验证结合活性。利用PD-1-his过夜包被，PBS清洗三遍，加入1％BSA/PBS，200uL/孔，37℃封闭1小时。PBS清洗三遍后加入100ul离心上清和不同质量的四价PD-L1混合液37℃孵育1小时，PBST清洗三遍后加入100ul 1:5000稀释的HRP偶联的山羊抗鼠IgG(Fab特异 性的)(Thermo)。PBST清洗三遍，加入100uL/孔TMB显色液，37℃显色10分钟，加入100uL/孔ELISA终止液，酶标仪读取OD450数值，以此验证阻断活性。选择ELISA读值低的克隆(即阻断作用强)进行测序获得抗体的序列，从中挑选阻断作用最强的6H6进入后续分析，6H6的抗体重链可变区和轻链可变区氨基酸序列分别为序列NO.7和NO.8。The candidate clones obtained from the primary screening were expanded and cultured with a 50ml shaker tube (thermo), and the periplasmic cavity extract was obtained by lysozyme (Shanghai Shenggong) combined with three freeze-thaw methods, and two ELISAs were used again to identify the binding and blocking activity. Coat with 50ng PD-1-his overnight, wash three times with PBS, add 1% BSA/PBS, 200uL/well, and block for 1 hour at 37°C. After washing three times with PBS, 100ul of the centrifuged supernatant was added and incubated at 37°C for 1 hour. After washing three times with PBST, 100ul of HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added at a 1:5000 dilution. Wash three times with PBST, add 100uL/well of TMB chromogenic solution, develop color at 37°C for 10 minutes, add 100uL/well of ELISA stop solution, and read the OD450 value with a microplate reader to verify the binding activity. Coat with PD-1-his overnight, wash with PBS three times, add 1% BSA/PBS, 200uL/well, block at 37°C for 1 hour. After washing three times with PBS, add 100ul centrifugation supernatant and tetravalent PD-L1 mixture of different quality and incubate at 37°C for 1 hour. After washing three times with PBST, add 100ul 1:5000 diluted HRP-conjugated goat anti-mouse IgG (Fab specific ) (Thermo). Wash three times with PBST, add 100uL/well TMB chromogenic solution, develop color at 37°C for 10 minutes, add 100uL/well ELISA stop solution, and read the OD450 value with a microplate reader to verify the blocking activity. Select the clone with low ELISA reading value (that is, strong blocking effect) for sequencing to obtain the sequence of the antibody, and select 6H6 with the strongest blocking effect for subsequent analysis. Sequence NO.7 and NO.8, respectively.
4.5表达6H6克隆hIgG1形式抗体4.5 Expression of 6H6 clone hIgG1 antibody
分别将6H6的重链可变区序列克隆至pFUSEss-CHIg-hG1中。分别将6H6轻链可变区序列克隆至pFUSE2ss-CLIg-hK中。分别以此双质粒1:1比例共转染293T细胞(ATCC)5天后，收集培养上清，用AKTA explorer 100(GE)纯化6H6抗体。6H6抗体经非还原SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约150k道尔顿。The heavy chain variable region sequences of 6H6 were cloned into pFUSEss-CHIg-hG1, respectively. The 6H6 light chain variable region sequences were cloned into pFUSE2ss-CLIg-hK, respectively. 293T cells (ATCC) were co-transfected with this two plasmids at a ratio of 1:1 for 5 days, the culture supernatant was collected, and the 6H6 antibody was purified with AKTA explorer 100 (GE). The 6H6 antibody was shown to be approximately 150 k Daltons in size by Coomassie brilliant blue staining after non-reducing SDS-PAGE electrophoresis.
4.6检测各hIgG1抗体(6H6及Nivolumab)分子水平阻断效果4.6 Detection of molecular level blocking effect of each hIgG1 antibody (6H6 and Nivolumab)
将6H6、Nivolumab与hIgG1同型对照(美国R&D Systems)一同进行竞争ELISA实验。简要步骤为：用每孔100ng的PD-1-huIgG1 Fc融合蛋白过夜包被，依次加入50ul 2ng/ul的不同的hIgG1抗体(6H6、Nivolumab和同型对照抗体)和50ul不同浓度的PD-L1-his(ACRO Biosystems)混合液，100ul 1:5000小鼠抗his标签-HRP(Thermo)显色以检测各抗体分子水平阻断效果。该检测步骤均含有三个复孔，ELISA结果的平均值详见表3。6H6分子水平阻断效果优于Nivolumab。6H6, Nivolumab and hIgG1 isotype control (American R&D Systems) were used for competition ELISA experiments. The brief steps are: overnight coating with 100ng of PD-1-huIgG1 Fc fusion protein per well, followed by adding 50ul 2ng/ul of different hIgG1 antibodies (6H6, Nivolumab and isotype control antibody) and 50ul of PD-L1- His (ACRO Biosystems) mixture, 100ul 1:5000 mouse anti-his tag-HRP (Thermo) color development to detect the blocking effect of each antibody molecule level. This detection step contains three duplicate wells, and the average value of ELISA results is shown in Table 3. The molecular level blocking effect of 6H6 is better than that of Nivolumab.
表3 6H6、Nivolumab与hIgG1同型对照抗体分子水平阻断效果Table 3 Molecular blocking effect of 6H6, Nivolumab and hIgG1 isotype control antibody
Figure PCTCN2021121785-appb-000003
Figure PCTCN2021121785-appb-000003
5.抗人PD-1鼠源抗体6H6的人源化5. Humanization of anti-human PD-1 murine antibody 6H6
鉴于6H6分子较优的分子水平阻断活性，针对6H6进行了人源化。首先开展基于互补决定区移植的抗体人源化，分别选择IGKV6-21*02和IGKJ4*01，IGHV3-23*04和IGHJ4*01人源胚系基因分别替换6H6对应的鼠源轻链和重链的胚系基因。其次我们预测出对抗体亲和力有贡献的关键氨基酸并完成回复突变：第一类氨基酸残基位于VL和VH结合界面上的残基， 对于两个结构域的Packing起到关键作用，第二类氨基酸残基靠近CDR区并且包埋于蛋白内部的残基，第三类氨基酸残基是与CDR区有直接相互作用的残基，包括疏水相互作用/氢键/盐桥等。基于上述规则构建三个候选分子，并通过体外亲和力测定优选出Hu_6H6分子作为人源化的6H6抗体版本，Hu_6H6的抗体重链可变区和轻链可变区氨基酸序列分别为序列NO.15和NO.16。In view of the superior molecular level blocking activity of the 6H6 molecule, humanization was carried out for 6H6. First carry out antibody humanization based on complementarity determining region transplantation, select IGKV6-21*02 and IGKJ4*01, IGHV3-23*04 and IGHJ4*01 human germline genes to replace the mouse light chain and heavy weight corresponding to 6H6, respectively Strand of germline genes. Secondly, we predict the key amino acids that contribute to antibody affinity and complete back mutation: the first type of amino acid residues are located at the binding interface of VL and VH, which play a key role in the packing of the two domains, and the second type of amino acid residues are located at the binding interface of VL and VH. The residues are close to the CDR region and are embedded in the interior of the protein. The third type of amino acid residues are residues that have direct interactions with the CDR region, including hydrophobic interactions/hydrogen bonds/salt bridges, etc. Based on the above rules, three candidate molecules were constructed, and the Hu_6H6 molecule was selected as the humanized 6H6 antibody version by in vitro affinity determination. The amino acid sequences of the antibody heavy chain variable region and light chain variable region of Hu_6H6 were sequence No. 15 and 1, respectively. NO.16.
实施例7：抗PD-1鼠抗的初步体外评价Example 7: Preliminary in vitro evaluation of anti-PD-1 mouse antibody
1.huIgG1型PD-1抗体体外中和试验1. In vitro neutralization test of huIgG1 PD-1 antibody
利用ELISA进行PD-1抗体体外中和试验，验证6H6和pembrolizumab的huIgG1型抗体阻断PD-L1与PD-1结合的能力：四度过夜包被PD-L1-huIgG1 Fc(ACRO Biosystems)1ug/ml 100ul每孔，洗板拍干后利用1％BSA in PBS 200ul/well 37℃孵育1小时进行封闭。0.1％PBST洗板3次拍干后用加入scFv-huIgG1 Fc型抗体和Biotinylated PD-1-huIgG1 Fc(Kactus Biosystems)混合物室温孵育1小时(2ug/ml 50ul的PD-1-huIgG1 Fc与从160ug/ml起始两倍梯度稀释的50ul抗体混合，共八个梯度)。0.1％PBST洗板3次，加入Streptavidin-HRP(R&D Systems)100ul每孔(1:200稀释)室温孵育1小时。0.1％PBST洗板6次后加入TMB显色液100ul每孔室温孵育10分钟，加入终止液100ul每孔后利用酶标仪进行OD450读数。对数据进行分析处理，计算得6H6h和pembrolizumab的huIgG1抗体对PD-1和PD-L1结合的半数抑制浓度IC50分别为3.605ug/ml和6.662ug/ml，如图2所示。In vitro neutralization test of PD-1 antibody by ELISA to verify the ability of huIgG1 type antibodies of 6H6 and pembrolizumab to block the binding of PD-L1 to PD-1: four times overnight coating of PD-L1-huIgG1 Fc (ACRO Biosystems) 1ug/ ml 100ul per well, wash the plate and pat dry, incubate with 1% BSA in PBS 200ul/well at 37°C for 1 hour for blocking. The plate was washed 3 times with 0.1% PBST, patted dry, and then incubated with a mixture of scFv-huIgG1 Fc-type antibody and Biotinylated PD-1-huIgG1 Fc (Kactus Biosystems) for 1 hour at room temperature (2ug/ml 50ul of PD-1-huIgG1 Fc with 160ug /ml initial two-fold serial dilution of 50ul antibody mix, a total of eight gradients). The plate was washed three times with 0.1% PBST, and 100ul of Streptavidin-HRP (R&D Systems) was added to each well (1:200 dilution) and incubated at room temperature for 1 hour. After washing the plate 6 times with 0.1% PBST, add 100 ul of TMB chromogenic solution to each well and incubate for 10 minutes at room temperature. After adding 100 ul of stop solution to each well, use a microplate reader to read the OD450. The data were analyzed and processed, and the median inhibitory concentration IC50 of 6H6h and pembrolizumab huIgG1 antibodies to PD-1 and PD-L1 binding were calculated to be 3.605ug/ml and 6.662ug/ml, respectively, as shown in Figure 2.
2.huIgG1型PD-1抗体体外结合试验2. In vitro binding test of huIgG1 PD-1 antibody
利用ELISA进行PD-1抗体亲和力测定，验证6H6和pembrolizumab的huIgG1抗体的亲和力：四度过夜包被PD-1 Protein,Human,Recombinant(His Tag)(北京义翘神州)0.5ug/ml 100ul每孔，洗板拍干后利用1％BSA in PBS 200ul/well 37℃孵育1小时进行封闭。0.1％PBST洗板3次拍干后用加入scFv-huIgG1 Fc型抗体室温孵育1小时(从10ug/ml起始两倍梯度稀释，共八个梯度)。0.1％PBST洗板3次，加入Goat anti-Mouse IgG F(ab')2Secondary Antibody,HRP(Thermo)/Goat Anti-Human IgG Secondary Antibody(HRP)(北京义翘神州)100ul每孔(1:10000稀释)室温孵育1小时。0.1％PBST洗板6次后加入TMB显色液100ul每孔室温孵育10分钟，加入终止液100ul每孔后利用酶标仪进行OD450读数。对数据进行分析处理，计算得6H6和pembrolizumab的huIgG1抗体与PD-1结合的EC50分别为0.01723ug/ml和0.01106ug/ml，如图3所示。ELISA was used to determine the affinity of PD-1 antibody to verify the affinity of huIgG1 antibody of 6H6 and pembrolizumab: four times overnight coating PD-1 Protein, Human, Recombinant (His Tag) (Beijing Yiqiao Shenzhou) 0.5ug/ml 100ul per well , wash the plate and pat dry, then use 1% BSA in PBS 200ul/well to incubate for 1 hour at 37°C for blocking. The plate was washed 3 times with 0.1% PBST, patted dry, and then incubated with scFv-huIgG1 Fc-type antibody for 1 hour at room temperature (two-fold serial dilution starting from 10ug/ml, a total of eight gradients). Wash the plate 3 times with 0.1% PBST, add Goat anti-Mouse IgG F(ab')2Secondary Antibody,HRP(Thermo)/Goat Anti-Human IgG Secondary Antibody(HRP)(Beijing Yiqiao Shenzhou) 100ul per well (1:10000 dilution) for 1 hour at room temperature. After washing the plate 6 times with 0.1% PBST, add 100 ul of TMB chromogenic solution to each well and incubate for 10 minutes at room temperature. After adding 100 ul of stop solution to each well, use a microplate reader to read the OD450. The data were analyzed and processed, and the calculated EC50s of the huIgG1 antibodies of 6H6 and pembrolizumab for binding to PD-1 were 0.01723ug/ml and 0.01106ug/ml, respectively, as shown in Figure 3.
实施例8：PD-1抗体刺激肿瘤浸润淋巴细胞(TIL)Example 8: PD-1 antibody stimulates tumor-infiltrating lymphocytes (TILs)
通过在肿瘤浸润淋巴细胞(TIL)的细胞培养基中加入PD-1抗体验证本申请抗体对TIL细 胞的刺激作用。复苏两个批次TIL细胞(供者A和供者B)，在含有IL-2的培养基中培养48小时。培养结束后清洗去除IL-2，以1E5/孔细胞加入到96孔板中，试验组(Transact+PD-1)根据说明书按照按1:1000的比例加入T Cell TransAct TM(Miltenyi Biotec)和1μg/ml的6H6 PD-1抗体刺激，对照组只加T Cell TransAct TM，孵育过夜后检测表达CD107a细胞比例及细胞因子分泌的结果。 The stimulating effect of the antibody of the present application on TIL cells was verified by adding PD-1 antibody to the cell culture medium of tumor infiltrating lymphocytes (TIL). Two batches of TIL cells (Donor A and Donor B) were resuscitated and cultured in IL-2-containing medium for 48 hours. After the culture, IL-2 was removed by washing, and 1E5/well cells were added to 96-well plates. The experimental group (Transact+PD-1) was added with T Cell TransAct TM (Miltenyi Biotec) and 1 μg according to the instructions at a ratio of 1:1000. /ml 6H6 PD-1 antibody stimulation, the control group only added T Cell TransAct TM , after overnight incubation, the proportion of cells expressing CD107a and the results of cytokine secretion were detected.
图4A显示的是在源于供者A的肿瘤浸润淋巴细胞(TIL)细胞培养基中加入PD-1抗体的CD107a细胞比例结果图。图4B显示的是在源于供者B的肿瘤浸润淋巴细胞(TIL)细胞培养基中加入PD-1抗体的CD107a细胞比例结果。结果显示，本申请的PD-1抗体可以提高表达CD107a的细胞比例。Figure 4A shows the results of the proportion of CD107a cells added to PD-1 antibody in the tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A. Figure 4B shows the results of the proportion of CD107a cells added to the PD-1 antibody in the tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor B. The results show that the PD-1 antibody of the present application can increase the proportion of cells expressing CD107a.
图5A-5F显示的是在源于供者A和供者B的肿瘤浸润淋巴细胞(TIL)细胞培养基中加入PD-1抗体的细胞因子IL-2、TNF-α和/或IFN-γ分泌结果图。结果显示，本申请的PD-1抗体可以提高IL-2、TNF-α和/或IFN-γ的分泌；本申请的PD-1抗体具有激活免疫细胞的能力。Figures 5A-5F show the cytokines IL-2, TNF-α and/or IFN-γ added to PD-1 antibody in tumor-infiltrating lymphocyte (TIL) cell culture medium derived from donor A and donor B Secretion results graph. The results show that the PD-1 antibody of the present application can increase the secretion of IL-2, TNF-α and/or IFN-γ; the PD-1 antibody of the present application has the ability to activate immune cells.
实施例9：混合淋巴细胞反应(MLR)实验检测PD-1抗体刺激T淋巴细胞Example 9: Mixed lymphocyte reaction (MLR) assay to detect PD-1 antibody to stimulate T lymphocytes
利用混合淋巴细胞反应(MLR)实验检测PD-1抗体能够刺激T淋巴细胞分泌IL-2及IFN-γ。将人PBMC调整细胞密度为2E7个/ml，采用CD14 MicroBeads,human(Miltenyi Biotec)分选单核细胞。添加50ng/ml GM-CSF(ACRO Biosystems)和50ng/ml IL-4(ACRO Biosystems)培养5天。补加10ng/ml的LPS(Sigma)和20ng/ml的TNF-α(ACRO Biosystems)诱导DC细胞成熟。使用Naive CD4+T Cell Isolation Kit II,human(Miltenyi Biotec)从不同人来源的PBMC中分离CD4+T细胞。96孔板中，每孔250μ1培养液含有1.0E5个分离的T细胞，2E4个诱导成熟的DC细胞以及一系列浓度梯度的pembrolizumab或本申请的PD-1抗体6H6。使用Human IgG1同型对照(Isotype control)作为阴性对照。混合淋巴细胞在37℃，5％CO 2细胞培养箱中培养3天，然后从96孔板中每孔取出100ul培养上清进行IL-2及IFN-γ测定。 The mixed lymphocyte reaction (MLR) assay was used to detect that PD-1 antibody could stimulate T lymphocytes to secrete IL-2 and IFN-γ. The cell density of human PBMC was adjusted to 2E7 cells/ml, and the monocytes were sorted by CD14 MicroBeads, human (Miltenyi Biotec). 50ng/ml GM-CSF (ACRO Biosystems) and 50ng/ml IL-4 (ACRO Biosystems) were added and cultured for 5 days. DC cell maturation was induced by supplementation of 10 ng/ml of LPS (Sigma) and 20 ng/ml of TNF-α (ACRO Biosystems). CD4+ T cells were isolated from PBMCs of various human origins using Naive CD4+ T Cell Isolation Kit II, human (Miltenyi Biotec). In a 96-well plate, 250 μl of culture medium per well contains 1.0E5 isolated T cells, 2E4 induced mature DC cells, and a series of concentration gradients of pembrolizumab or the PD-1 antibody 6H6 of the present application. A Human IgGl Isotype control was used as a negative control. The mixed lymphocytes were cultured in a 37°C, 5% CO2 cell incubator for 3 days, and then 100ul of the culture supernatant was removed from each well of the 96-well plate for IL-2 and IFN-γ assays.
图6A-6B显示的是混合淋巴细胞反应(MLR)实验检测PD-1抗体对于T淋巴细胞分泌IL-2及IFN-γ的刺激结果图。结果显示，本申请的PD-1抗体具有激活免疫细胞的能力。Figures 6A-6B show the results of the mixed lymphocyte reaction (MLR) assay to detect the stimulation results of PD-1 antibody on the secretion of IL-2 and IFN-γ by T lymphocytes. The results show that the PD-1 antibody of the present application has the ability to activate immune cells.
实施例10：PD-1抗体增强免疫细胞的杀伤能力Example 10: PD-1 antibody enhances the killing ability of immune cells
通过在NYESO-1TCR-T细胞对A375细胞的杀伤中加入PD-1抗体验证PD-1抗体是否增强TCR-T杀伤能力。从新鲜分离的人PBMC细胞中经CD3MicroBeads,human(Miltenyi Biotec130-050-101)分选出CD3阳性T细胞。可以通过任意的转导方法获得TCR-T细胞，例如将携带NY-ESO-1TCR(Kite Pharma)慢病毒用于转导分选出的CD3阳性T细胞，流式检测转导效率可以为40％。A375细胞每孔2E4细胞铺板到96孔板中，加入2E4/孔转导后的 NYESO-1TCR细胞，同时加入10μg/ml的6H6抗体及对照抗体pembrolizumab作为试验组，A375细胞中加入等量PBMC细胞作为阴性对照，只有A375细胞作为空白对照，所有组每孔同时加入荧光染料SuperView TM 488 Caspase-3。将96孔板转入到IncuCyte S3每3小时自动拍照33小时后统计分析杀伤结果。 It was verified whether PD-1 antibody enhanced the killing ability of TCR-T by adding PD-1 antibody to the killing of A375 cells by NYESO-1 TCR-T cells. CD3 positive T cells were sorted from freshly isolated human PBMC cells by CD3 MicroBeads, human (Miltenyi Biotec 130-050-101). TCR-T cells can be obtained by any transduction method, for example, lentivirus carrying NY-ESO-1TCR (Kite Pharma) is used to transduce the sorted CD3-positive T cells, and the transduction efficiency can be 40% by flow detection . A375 cells were plated in 96-well plates with 2E4 cells per well, NYESO-1TCR cells transduced with 2E4/well were added, and 10 μg/ml of 6H6 antibody and control antibody pembrolizumab were added as the experimental group, and the same amount of PBMC cells were added to the A375 cells As a negative control, only A375 cells were used as a blank control, and the fluorescent dye SuperView TM 488 Caspase-3 was added to each well of all groups at the same time. The 96-well plate was transferred to IncuCyte S3 every 3 hours, and pictures were taken automatically for 33 hours, and the killing results were statistically analyzed after 33 hours.
图7显示的是加入PD-1抗体对TCR-T细胞的杀伤能力增强结果图。结果显示本申请的PD-1抗体可以增强免疫细胞的杀伤能力。Figure 7 shows the results of the enhanced killing ability of TCR-T cells by adding PD-1 antibody. The results show that the PD-1 antibody of the present application can enhance the killing ability of immune cells.
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。The foregoing detailed description has been presented by way of explanation and example, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments presently enumerated in this application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims (37)

  1. 分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:7或9所示的氨基酸序列。An isolated antigen binding protein comprising at least one CDR in the VH of an antibody heavy chain variable region, the VH comprising the amino acid sequence shown in SEQ ID NO: 7 or 9.
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。The isolated antigen-binding protein of claim 1, which comprises an antibody or antigen-binding fragment thereof.
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。 The isolated antigen-binding protein of any one of claims 1-2, wherein the antigen-binding fragment comprises a Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb .
  4. 根据权利要求2-3中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。The isolated antigen binding protein of any one of claims 2-3, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其与参比抗体竞争结合PD-1，其中所述参比抗体包含轻链可变区和重链可变区，所述参比抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:4所示的氨基酸序列；所述LCDR2包含SEQ ID NO:5所示的氨基酸序列；所述LCDR3包含SEQ ID NO:6所示的氨基酸序列，所述参比抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:1所示的氨基酸序列；所述HCDR2包含SEQ ID NO:2所示的氨基酸序列；所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。The isolated antigen-binding protein of any one of claims 1-4, which competes with a reference antibody for binding to PD-1, wherein the reference antibody comprises a light chain variable region and a heavy chain variable region, wherein the The light chain variable region of the reference antibody comprises LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5; the LCDR3 Comprising the amino acid sequence shown in SEQ ID NO: 6, the heavy chain variable region of the reference antibody comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 comprises SEQ ID NO: 1 The amino acid sequence shown in ID NO: 2; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3.
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:3所示的氨基酸序列。The isolated antigen binding protein of any one of claims 1-5, the VH comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
  7. 根据权利要求6所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。The isolated antigen binding protein of claim 6, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2.
  8. 根据权利要求6-7中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:1所示的氨基酸序列。The isolated antigen binding protein of any one of claims 6-7, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1.
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，所述VH包含HFR1，HFR2，HFR3和HFR4，其中所述HFR1包含SEQ ID NO:11或SEQ ID NO:19所示的氨基酸序列。The isolated antigen binding protein of any one of claims 1-8, wherein the VH comprises HFR1, HFR2, HFR3 and HFR4, wherein the HFR1 comprises SEQ ID NO: 11 or SEQ ID NO: 19 amino acid sequence.
  10. 根据权利要求9中任一项所述的分离的抗原结合蛋白，其中所述HFR2包含SEQ ID NO:12或SEQ ID NO:20所示的氨基酸序列。The isolated antigen-binding protein of any one of claims 9, wherein the HFR2 comprises the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 20.
  11. 根据权利要求9-10中任一项所述的分离的抗原结合蛋白，其中所述HFR3包含SEQ ID NO:13或SEQ ID NO:21所示的氨基酸序列。The isolated antigen binding protein of any one of claims 9-10, wherein the HFR3 comprises the amino acid sequence set forth in SEQ ID NO:13 or SEQ ID NO:21.
  12. 根据权利要求9-11中任一项所述的分离的抗原结合蛋白，其中所述HFR4包含SEQ ID NO:14或SEQ ID NO:22所示的氨基酸序列。The isolated antigen binding protein of any one of claims 9-11, wherein the HFR4 comprises the amino acid sequence set forth in SEQ ID NO:14 or SEQ ID NO:22.
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其包含VH，且所述VH包含 SEQ ID NO:7或9所示的氨基酸序列。The isolated antigen-binding protein of any one of claims 1-12, comprising a VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 7 or 9.
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区。The isolated antigen binding protein of any one of claims 1-13, comprising an antibody heavy chain constant region.
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG恒定区。The isolated antigen binding protein of claim 14, wherein the heavy chain constant region is derived from a human IgG constant region.
  16. 根据权利要求14-15中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。The isolated antigen binding protein of any one of claims 14-15, wherein the heavy chain constant region is derived from a human IgG4 constant region and/or a human IgGl constant region.
  17. 根据权利要求1-16中任一项所述的分离的抗原结合蛋白，其包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:8或10所示的氨基酸序列。The isolated antigen-binding protein of any one of claims 1-16, comprising at least one CDR in the variable region VL of an antibody light chain, the VL comprising the amino acid sequence shown in SEQ ID NO: 8 or 10 .
  18. 根据权利要求17所述的分离的抗原结合蛋白，所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:4所示的氨基酸序列。The isolated antigen binding protein of claim 17, wherein the VL comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4.
  19. 根据权利要求18所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。The isolated antigen binding protein of claim 18, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5.
  20. 根据权利要求18-19中任一项所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:6所示的氨基酸序列。The isolated antigen binding protein of any one of claims 18-19, wherein the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6.
  21. 根据权利要求17-20中任一项所述的分离的抗原结合蛋白，所述VL包含LFR1，LFR2，LFR3和LFR4，其中所述LFR1包含SEQ ID NO:15或SEQ ID NO:23所示的氨基酸序列。The isolated antigen binding protein of any one of claims 17-20, the VL comprising LFR1, LFR2, LFR3 and LFR4, wherein the LFR1 comprises the set forth in SEQ ID NO:15 or SEQ ID NO:23 amino acid sequence.
  22. 根据权利要求21中任一项所述的分离的抗原结合蛋白，其中所述LFR2包含SEQ ID NO:16或SEQ ID NO:24所示的氨基酸序列。The isolated antigen-binding protein of any one of claims 21, wherein the LFR2 comprises the amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO: 24.
  23. 根据权利要求21-22中任一项所述的分离的抗原结合蛋白，其中所述LFR3包含SEQ ID NO:17或SEQ ID NO:25所示的氨基酸序列。The isolated antigen binding protein of any one of claims 21-22, wherein the LFR3 comprises the amino acid sequence set forth in SEQ ID NO:17 or SEQ ID NO:25.
  24. 根据权利要求21-23中任一项所述的分离的抗原结合蛋白，其中所述LFR4包含SEQ ID NO:18或SEQ ID NO:26所示的氨基酸序列。The isolated antigen binding protein of any one of claims 21-23, wherein the LFR4 comprises the amino acid sequence set forth in SEQ ID NO:18 or SEQ ID NO:26.
  25. 根据权利要求1-24中任一项所述的分离的抗原结合蛋白，其包含VL，且所述VL包含SEQ ID NO:8或10所示的氨基酸序列。The isolated antigen-binding protein of any one of claims 1-24, comprising a VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 8 or 10.
  26. 根据权利要求1-25中任一项所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：The isolated antigen-binding protein of any one of claims 1-25 having one or more of the following properties:
    a)能够结合人的PD-1；a) capable of binding to human PD-1;
    b)能够阻断PD-1和PD-L1结合；b) Can block the binding of PD-1 and PD-L1;
    c)能够阻断PD-1和PD-L2结合；c) can block the binding of PD-1 and PD-L2;
    d)能够刺激免疫细胞中IL-2、TNF-α和/或IFN-γ的分泌；d) can stimulate the secretion of IL-2, TNF-α and/or IFN-γ in immune cells;
    e)能够抑制肿瘤生长和/或肿瘤细胞增殖；e) capable of inhibiting tumor growth and/or tumor cell proliferation;
    f)能够提高免疫细胞的杀伤能力。f) can improve the killing ability of immune cells.
  27. 分离的一种或多种核酸分子，其编码权利要求1-26中任一项所述的分离的抗原结合蛋白。Isolated one or more nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-26.
  28. 载体，其包含根据权利要求27所述的核酸分子。A vector comprising the nucleic acid molecule of claim 27.
  29. 细胞，其包含根据权利要求27所述的核酸分子或根据权利要求28所述的载体。A cell comprising the nucleic acid molecule of claim 27 or the vector of claim 28.
  30. 制备权利要求1-26中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-26中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求29所述的细胞。A method for preparing the isolated antigen-binding protein of any one of claims 1-26, the method comprising culturing the isolated antigen-binding protein of any one of claims 1-26 under conditions that allow expression of the isolated antigen-binding protein The cell of claim 29.
  31. 药物组合物，其包含权利要求1-26中任一项所述的分离的抗原结合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体和/或权利要求29所述的细胞，以及任选地药学上可接受的佐剂。A pharmaceutical composition comprising the isolated antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the carrier of claim 28 and/or the cell of claim 29 , and optionally a pharmaceutically acceptable adjuvant.
  32. 权利要求1-26中任一项所述的分离的抗原结合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞和/或权利要求31所述的药物组合物在制备药物中的用途，所述药物用于治疗PD-1介导的疾病或病症。The isolated antigen binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, the vector of claim 28, the cell of claim 29 and/or the claim 31 Use of the pharmaceutical composition in the preparation of a medicament for treating a disease or condition mediated by PD-1.
  33. 根据权利要求32所述的用途，其中所述PD-1介导的疾病或病症包括癌症。The use of claim 32, wherein the PD-1 mediated disease or disorder comprises cancer.
  34. 抑制PD-1与PD-L1结合的方法，其包括向有需要的受试者施用有效量的权利要求1-26中任一项所述的抗原结合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞和/或权利要求31所述的药物组合物。A method for inhibiting the binding of PD-1 to PD-L1, comprising administering to a subject in need an effective amount of the antigen-binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, The vector of claim 28, the cell of claim 29 and/or the pharmaceutical composition of claim 31.
  35. 抑制PD-1与PD-L2结合的方法，其包括向有需要的受试者施用有效量的权利要求1-26中任一项所述的抗原结合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞和/或权利要求31所述的药物组合物。A method for inhibiting the combination of PD-1 and PD-L2, comprising administering to a subject in need an effective amount of the antigen-binding protein of any one of claims 1-26, the nucleic acid molecule of claim 27, The vector of claim 28, the cell of claim 29 and/or the pharmaceutical composition of claim 31.
  36. 预防、缓解或治疗PD-1介导的疾病或病症的方法，其包括向有需要的受试者施用有效量的权利要求1-26中任一项所述的抗原结合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞和/或权利要求31所述的药物组合物。A method for preventing, alleviating or treating a PD-1-mediated disease or disorder, comprising administering to a subject in need an effective amount of the antigen-binding protein of any one of claims 1-26, the antigen-binding protein of claim 27 the nucleic acid molecule, the vector of claim 28, the cell of claim 29, and/or the pharmaceutical composition of claim 31.
  37. 根据权利要求36所述的方法，其中所述PD-1介导的疾病或病症包括癌症。The method of claim 36, wherein the PD-1 mediated disease or disorder comprises cancer.
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