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WO2019184909A1 - 新型抗体分子、其制备方法及其用途 - Google Patents

新型抗体分子、其制备方法及其用途 Download PDF

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Publication number
WO2019184909A1
WO2019184909A1 PCT/CN2019/079671 CN2019079671W WO2019184909A1 WO 2019184909 A1 WO2019184909 A1 WO 2019184909A1 CN 2019079671 W CN2019079671 W CN 2019079671W WO 2019184909 A1 WO2019184909 A1 WO 2019184909A1
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domain
immunoglobulin
antibody
polypeptide chain
terminus
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PCT/CN2019/079671
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English (en)
French (fr)
Inventor
刘军建
缪小牛
匡智慧
Original Assignee
信达生物制药(苏州)有限公司
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Priority claimed from CN201910196438.4A external-priority patent/CN110305210B/zh
Application filed by 信达生物制药(苏州)有限公司 filed Critical 信达生物制药(苏州)有限公司
Priority to US16/965,229 priority Critical patent/US11746148B2/en
Priority to EP19775977.2A priority patent/EP3778648A4/en
Priority to JP2020545187A priority patent/JP7438958B2/ja
Publication of WO2019184909A1 publication Critical patent/WO2019184909A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention generally relates to the field of immunology and antibody engineering.
  • the invention relates to a novel novel engineered antibody molecule, a polynucleotide encoding the antibody molecule, a vector comprising the polynucleotide, a host cell comprising the polynucleotide or vector, comprising Immunoconjugates and pharmaceutical compositions of the antibody molecules, and uses of the antibody molecules for immunotherapy, prevention, and/or diagnosis of a disease.
  • Antibody molecules are capable of targeted and specific binding to their corresponding antigens, and are increasingly becoming important therapeutic and prophylactic agents for various diseases (eg, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.). / or diagnostic agent.
  • diseases eg, cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.
  • diagnostic agent e.g., cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.
  • monospecific antibodies directed against only one target have some limitations in clinical applications. Patients may develop resistance or no response after receiving monospecific antibody therapy.
  • multispecific antibodies are capable of specifically binding to different antigens, when one antigen is located on a particular immune cell and the other antigen is on a disease cell, a multispecific antibody (eg, bispecific) Sexual antibodies) can redirect specific immune cells to diseased cells to enhance the lethality of immune cells to diseased cells.
  • multispecific antibodies eg, bispecific antibodies
  • Blinatumomab is the first single-chain bispecific antibody with a molecular weight of approximately 55 kDa for the treatment of B-cell non-Hodgkin's lymphoma (NHL) and B precursor acute lymphoblastic leukemia (ALL).
  • NHL B-cell non-Hodgkin's lymphoma
  • ALL B precursor acute lymphoblastic leukemia
  • the two single-chain Fv molecules directed against the CD19 molecule and against the CD3 molecule are fused by a flexible linker peptide, which utilizes CD19 expressed in almost all B lymphocyte tumors and CD3 expressed on T cells, making T
  • the cells are tightly linked to the target cells (tumor cells), which release perforin and telomerase into the synaptic cleft, causing a series of chemical reactions in the tumor cells, thereby destroying the tumor cells (Nagorsen D. and Baeuerle PA, Immunomodulatory) Therapy of cancer with T cell-engaging BiTE antibody blinatumomab, Exp Cell Res, 2011, 317: 1255-1260).
  • Catumaxomab is a chimera consisting of two half-antibodies derived from the parental mouse IgG2a isotype and the rat IgG2b isotype, each having a light chain and a heavy chain, anti-CD3 rat IgG2b half.
  • Antibodies for T cell recognition mouse IgG2a half antibodies against tumor cell surface antigen EpCAM (epithelial adhesion molecule) for tumor cell recognition (Chelius D et al, Structural and functional characterization of the trifunctional antibody catumaxomab, MAbs, 2010, 2:309-319).
  • Catumaxomab Approved in Europe in April 2009 for the treatment of malignant ascites caused by EpCAM-positive epithelial-derived metastases.
  • Multispecific antibodies can be divided into many classes depending on the components and the manner in which they are constructed. For example, according to the left and right basic symmetry of the multispecific antibody structure, it can be divided into a symmetric structure and an asymmetric structure; according to the Fc region of the multispecific antibody with or without IgG, it can be divided into an antibody pattern having an Fc region and an Fc-free region.
  • the antibody pattern according to the number of antigen binding sites in the multispecific antibody, it can be classified into divalent, trivalent, tetravalent or higher valence antibodies and the like.
  • Blinatumomab can be produced by large-scale culture of recombinant Chinese hamster ovary (CHO) cells, it is easy to form aggregates and has a short half-life in vivo. In actual use, an additional infusion set is required; the Catumaxomab production process is complicated and the mouse heterologous antibody is more susceptible to immunogenicity in the human body.
  • CHO Chinese hamster ovary
  • the present invention provides a novel multispecific antibody format by using a single domain antigen binding site with a small molecular weight and high stability as a building block, with the N-terminus or C-terminus of the Fab fragment. After ligation, the resulting adaptor is ligated to the Fc region, and is easily expressed in cultured cells in vitro without complicated production processes; in addition, the presence of the Fc region in the antibody pattern of the present invention allows expression of the present invention in cultured cells.
  • Purified antibodies can be obtained using single-step affinity chromatography, and the antibodies of the invention have longer serum half-life in vivo and can elicit effector functions, such as antibody-dependent cell-mediated cells.
  • the multispecific antibody pattern of the present invention is capable of maintaining the affinity of each antigen binding site in the multispecific antibody to bind to a corresponding different epitope, and does not cause steric hindrance when binding different epitopes. Interference, with good drug-forming properties. Further, the multispecific antibody format of the invention is physically stable and biologically stable, which allows the antibody to be more productive and developable.
  • the antibody molecule is capable of binding to one or more antigens with high affinity and high specificity, preferably to two or more antigens.
  • the invention also provides nucleic acid molecules encoding the antibody molecules, expression vectors, host cells and methods for producing the antibody molecules.
  • the invention also provides immunoconjugates and pharmaceutical compositions comprising the antibody molecules of the invention.
  • the antibody molecules disclosed herein can be used alone or in combination with other drugs or other therapeutic modalities for the treatment, prevention, and/or diagnosis of diseases such as autoimmune diseases, acute and chronic inflammatory diseases, infectious diseases (eg, chronic infectious diseases or sepsis). ), tumors, etc.
  • the invention provides an antibody molecule having one or more of the following properties:
  • an antibody molecule of the invention comprises (i) a single domain antigen binding site; (ii) an antigen binding Fab fragment; wherein said (i) is located in said (ii) light chain variable structure The N-terminus of the domain (VL) or the C-terminus of the light chain constant region (CL), or the (i) N-terminal or immunoglobulin CH1 structure of the heavy chain variable domain (VH) of (ii) C-terminus of the domain, and said (i) and (ii) respectively bind the same or different antigens, with or without a linker peptide between (i) and (ii); and at (i) and Iii) The C-terminal (iii) immunoglobulin Fc domain.
  • the antibody molecule of the invention comprises at least four antigen binding sites, at least two single domain antigen binding sites and an antigen binding site in at least two Fab fragments, respectively, in combination with at least four, Three, two different antigens, or one antigen.
  • the antigen binding site in the antibody molecule of the invention binds to the same or different epitopes in the antigen molecule.
  • an antibody molecule of the invention comprises four antigen binding sites, wherein two single domain antigen binding sites bind to the same or different epitopes in the first antigen, and two Fab fragments bind to the second antigen The same or different epitopes, the first antigen being different from the second antigen.
  • a glycine and/or serine residue used alone or in combination between (i) and (ii) in the antibody molecule of the present invention is used as a linker peptide, for example, the linker peptide comprises an amino acid sequence (Gly 4 Ser)n, where n is a positive integer equal to or greater than 1, for example, n is a positive integer in 1-7, for example, n is 2, 3, 4, 5, 6.
  • the single domain antigen binding site in the antibody molecule of the invention is selected from the group consisting of a heavy chain variable domain (VH), a light chain variable domain (VL), and a heavy chain of an antibody that naturally lacks a light chain.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a variable domain for example, a heavy chain variable domain of a naturally occurring heavy chain antibody in a Camelidae species
  • NAR new antigen receptor
  • fish eg, VH-like single domains in naturally occurring IgNAR in shark serum
  • recombinant single domain antigen binding sites derived from them eg, camelized human VH domain, humanized camelid antibody heavy Chain variable domain.
  • the single domain antigen binding site in an antibody molecule of the invention is selected from the heavy chain variable domain of a naturally occurring heavy chain antibody in camelid species, a camelized human VH domain And humanized camelid antibody heavy chain variable domains.
  • the heavy chain variable domain derived from a heavy chain antibody that naturally lacks the light chain is also referred to herein simply as VHH to distinguish it from the conventional VH of a four-chain immunoglobulin.
  • VHH molecules can be derived from antibodies produced in camelid species such as camels, alpacas, dromedaries, llamas and guanaco. Other species other than camelids can also produce heavy chain antibodies that naturally lack light chains, and such VHHs are also within the scope of the invention.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light chain and is located in the immunization A N-terminal single domain antigen binding site of a globin light chain variable domain (VL), such as VHH; each polypeptide chain in the second polypeptide chain and the fourth polypeptide chain comprises an immunoglobulin heavy chain.
  • VL globin light chain variable domain
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light chain and is located in the immunization A single domain antigen binding site at the C-terminus of the globose light chain constant region (CL), such as VHH; each polypeptide chain in the second polypeptide chain and the fourth polypeptide chain comprises an immunoglobulin heavy chain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each polypeptide chain in the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light chain; a second polypeptide Each polypeptide chain in the chain and the fourth polypeptide chain comprises an immunoglobulin heavy chain and a single domain antigen binding site at the N-terminus of the immunoglobulin heavy chain, such as VHH.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each polypeptide chain in the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light chain; a second polypeptide Each polypeptide chain in the chain and the fourth polypeptide chain comprises an immunoglobulin heavy chain variable region, an immunoglobulin CH1 domain, a single domain antigen binding site (eg, VHH), immunization from the N-terminus to the C-terminus. Globulin CH2, CH3 and optionally CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises a single domain antigen from the N-terminus to the C-terminus Binding site (eg VHH), immunoglobulin heavy chain variable domain (VH) and immunoglobulin light chain constant region (CL); each polypeptide chain in the second polypeptide chain and the fourth polypeptide chain
  • the immunoglobulin light chain variable domain (VL), immunoglobulin CH1, CH2, CH3 and optionally the CH4 domain are comprised from the N-terminus to the C-terminus.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin heavy from the N-terminus to the C-terminus a chain variable domain (VH), an immunoglobulin light chain constant region (CL), and a single domain antigen binding site (eg, VHH); each polypeptide chain in the second polypeptide chain and the fourth polypeptide chain
  • the immunoglobulin light chain variable domain (VL), immunoglobulin CH1, CH2, CH3 and optionally the CH4 domain are comprised from the N-terminus to the C-terminus.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin heavy from the N-terminus to the C-terminus a chain variable domain (VH) and an immunoglobulin light chain constant region (CL); each polypeptide chain in the second polypeptide chain and the fourth polypeptide chain comprises a single domain antigen binding from the N-terminus to the C-terminus Site (eg, VHH), immunoglobulin light chain variable domain (VL), immunoglobulin CH1, CH2, CH3, and optionally CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin heavy from the N-terminus to the C-terminus a chain variable domain (VH) and an immunoglobulin light chain constant region (CL); each of the second polypeptide chain and the fourth polypeptide chain comprises an immunoglobulin light chain from the N-terminus to the C-terminus Variable domain (VL), immunoglobulin CH1 domain, single domain antigen binding site (eg, VHH), immunoglobulin CH2, CH3, and optionally CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises a single domain antigen from the N-terminus to the C-terminus a binding site (eg, VHH), an immunoglobulin light chain variable domain (VL), and an immunoglobulin CH1 domain; each of the second polypeptide chain and the fourth polypeptide chain is from the N-terminus to The C-terminus comprises an immunoglobulin heavy chain variable domain (VH), an immunoglobulin light chain constant region (CL), an immunoglobulin CH2, CH3, and optionally a CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light from the N-terminus to the C-terminus a chain variable domain (VL), an immunoglobulin CH1 domain, and a single domain antigen binding site (eg, VHH); each of the second polypeptide chain and the fourth polypeptide chain is from the N terminus to The C-terminus comprises an immunoglobulin heavy chain variable domain (VH), an immunoglobulin light chain constant region (CL), an immunoglobulin CH2, CH3, and optionally a CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light from the N-terminus to the C-terminus a chain variable domain (VL) and an immunoglobulin CH1 domain; each of the second polypeptide chain and the fourth polypeptide chain comprises a single domain antigen binding site from the N-terminus to the C-terminus (eg VHH), immunoglobulin heavy chain variable domain (VH), immunoglobulin light chain constant region (CL), immunoglobulin CH2, CH3 and optionally CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the invention provides an antibody molecule comprising four polypeptide chains, wherein each of the first polypeptide chain and the third polypeptide chain comprises an immunoglobulin light from the N-terminus to the C-terminus a chain variable domain (VL) and an immunoglobulin CH1 domain; each of the second polypeptide chain and the fourth polypeptide chain comprises an immunoglobulin heavy chain variable domain from the N-terminus to the C-terminus (VH), immunoglobulin light chain constant region (CL), single domain antigen binding site (eg, VHH), immunoglobulin CH2, CH3, and optionally CH4 domain.
  • the immunoglobulin is an IgG1, IgG2 or IgG4 immunoglobulin, more preferably the immunoglobulin is a human IgG1 immunoglobulin.
  • the inventors have also designed amino acid residues capable of stabilizing the molecular structure of the antibody and facilitating proper coupling or pairing between the individual strands.
  • a hinge region having a "CPPC" amino acid residue is included in an Fc domain of a second polypeptide chain and a fourth polypeptide chain of an antibody molecule such that the second polypeptide chain and the fourth polypeptide chain pass each other
  • the disulfide bond formed between the amino acid residues at the hinge region is stably associated.
  • the second polypeptide chain and the fourth polypeptide chain of the antibody molecule of the invention comprise Y349C and S354C, respectively, in the respective Fc domains or S354C and Y349C, respectively (according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), the EU index is numbered, hereinafter referred to as "EU number"), whereby the second polypeptide chain and the fourth polypeptide The strand further forms an interchain disulfide bond in the Fc region to stabilize the correct pairing of the second polypeptide chain and the fourth polypeptide chain.
  • the second polypeptide chain and/or the fourth polypeptide chain of an antibody molecule of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the amino acid mutation is present in the CH2 domain of the Fc region, eg, the antibody molecule is comprised at positions 234 and 235 (EU numbering) of the second polypeptide chain and/or the fourth polypeptide chain Amino acid substitution.
  • the amino acid substitutions are L234A and L235A (hereinafter referred to as "LALA mutations").
  • the Fc domain of each of the second polypeptide chain and the fourth polypeptide chain of the antibody molecule of the invention comprises a bulge ("knob") or a hole ("hole”, respectively). And said bulges or vacancies in the Fc domain of the second polypeptide chain can be placed in said holes or bulges in the Fc domain of the fourth polypeptide chain, respectively, whereby said second The peptide chain and the fourth polypeptide chain form a "knob-in-hole" stable association with each other.
  • an amino acid substitution T366W is included in one of the second polypeptide chain and the fourth polypeptide chain, and is included in the other of the second polypeptide chain and the fourth polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering).
  • the protrusions in one strand can be placed in the cavities in the other strand, facilitating the correct pairing of the second polypeptide chain and the fourth polypeptide chain.
  • the first polypeptide chain of the antibody molecule of the invention and the immunoglobulin CL domain and the CH1 domain of the second polypeptide chain comprise a bulge or a hole, respectively, and said The protrusions or holes may be placed in the holes or protrusions in the CL domain, respectively, such that the first polypeptide chain and the second polypeptide chain also form a stable association of "binding" to each other.
  • the third polypeptide chain of the antibody molecule of the present invention and the immunoglobulin CL domain and the CH1 domain of the fourth polypeptide chain also respectively contain protrusions or holes, and the protrusions in the CH1 domain Or holes may be placed in the holes or bulges in the CL domain, respectively, such that the third polypeptide chain and the fourth polypeptide chain also form a stable association of "binding" to each other.
  • two single domain antigen binding sites in an antibody molecule of the invention bind to the same epitope in a first antigen, and two Fab fragments bind to the same epitope in a second antigen, whereby The antibody molecule is a bispecific antibody directed against the first antigen and the second antigen.
  • the type of antigen to which the antibody molecule of the present invention specifically binds is not particularly limited, and the antigen may be, for example, a cytokine, a growth factor, a hormone, a signaling protein, an inflammatory mediator, a ligand, a cell surface receptor, or a fragment thereof.
  • the antigen to which the antibody molecule of the invention specifically binds is selected from the group consisting of a tumor-associated antigen, an immunological checkpoint molecule, an angiogenesis-inducing factor, a member of the tumor necrosis factor receptor superfamily, and a costimulatory molecule in the immune system, and Ligands and/or receptors for these molecules, for example, OX40, CD47, PD1, PD-L1, PD-L2, LAG-3, 4-1BB (CD137), VEGF, and GITR.
  • the present invention exemplifies several bispecific antibodies as described below.
  • the antibody molecule of the invention is an anti-OX40/PD-L1 bispecific antibody capable of being at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably An affinity constant of about 10 9 M -1 or greater binds to OX40, a member of the tumor necrosis factor (TNF) receptor family expressed on the surface of lymphocytes, thereby activating T cells, for example, enhancing the immune stimulation/effector function of effector T cells and / or allowing these cells to proliferate and / or downregulate the immunosuppressive function of regulatory T cells; and at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger
  • the affinity constant binds to PD-L1 on the surface of tumor cells, thereby inhibiting the binding of PD-1 on T cells to PD-L1 on the surface of tumor cells, inducing T cell activation and exerting an anti-tumor effect.
  • the anti-OX40/PD-L1 bispecific antibody of the invention consists of four polypeptide chains that are substantially symmetric about the left and right, wherein two polypeptide chains in the left half and two polypeptide chains in the right half Each comprising (i) a single domain antigen binding site; (ii) an antigen binding Fab fragment; wherein said (i) is located at the N-terminus of said (ii) light chain variable domain (VL) or The C-terminus of the light chain constant region (CL), or the (i) the N-terminus of the heavy chain variable domain (VH) of the (ii) or the C-terminus of the immunoglobulin CH1 domain, and (i), (ii) binding to OX40 or PD-L1, respectively, with or without a linking peptide between (i) and (ii); and (iii) at the C-terminus of (i) and (ii) Immunoglobulin Fc domain.
  • the single domain antigen binding site in the anti-OX40/PD-L1 bispecific antibody of the invention is a VHH that specifically binds to PD-L1, and the Fab fragment is an anti-OX40 antibody Fab that specifically binds OX40 Fragment.
  • the VHH that specifically binds to PD-L1 in the anti-OX40/PD-L1 bispecific antibody of the invention comprises CDR1, SEQ ID NO: 4 set forth in SEQ ID NO: CDR2 shown as CDR2 and SEQ ID NO: 5, or one, two, three, four, five, six or more amino acids with one or more of the three CDRs Sequences that vary (eg, amino acid substitutions or deletions);
  • the anti-OX40 antibody Fab fragment that specifically binds OX40 in an anti-OX40/PD-L1 bispecific antibody of the invention comprises a SEQ derived from the anti-OX40 antibody ADI-20112 ID NO: all 6 heavy chain complementarity determining regions (CDRs) and light chain CDRs in the paired heavy chain variable region sequence/light chain variable region sequence shown in 11/7, or in the 6 CDRs
  • One or more CDRs have a sequence of one, two, three, four, five, six or more amino acid changes (eg, amino acid substitution
  • the VHH that specifically binds to PD-L1 in the anti-OX40/PD-L1 bispecific antibody of the invention comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, Or a sequence substantially identical thereto (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical); the anti-OX40/PD-L1 of the present invention
  • the anti-OX40 antibody Fab fragment that specifically binds OX40 in the bispecific antibody comprises the paired heavy chain variable region sequence/light chain shown in SEQ ID NO: 11/7 derived from the anti-OX40 antibody ADI-20112
  • the variable region sequence or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 with the paired heavy chain variable region sequence/light chain variable region sequence %, 99% or more sequences of sequence identity.
  • the anti-OX40/PD-L1 bispecific antibody of the invention consists of four polypeptide chains substantially symmetric about each other, wherein the two polypeptide chains of the left half of the antibody molecule comprise a first polypeptide chain of SEQ ID NO: 6 and a second polypeptide chain of SEQ ID NO: 10; comprising a first polypeptide chain of SEQ ID NO: 14 and SEQ ID NO: 10, respectively a second polypeptide chain; comprising a first polypeptide chain represented by SEQ ID NO: 15 and a second polypeptide chain represented by SEQ ID NO: 16; respectively comprising the first one represented by SEQ ID NO: 15 a polypeptide chain and a second polypeptide chain of SEQ ID NO: 17; or substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, a sequence of 98%, 99% or higher identical; accordingly, wherein the two polypeptide chains of the right half of the antibody molecule comprise a first polypeptide chain
  • the antibody molecule of the invention is an anti-VEGF/GITR bispecific antibody capable of being at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger affinity constant binds to Vascular Endothelial Cell Growth Factor (VEGF), thereby blocking the binding of VEGF to its receptor VEGFR, making VEGFR unable to activate, thereby exerting anti-angiogenic effects.
  • VEGF Vascular Endothelial Cell Growth Factor
  • an anti-tumor angiogenesis effect inhibiting tumor growth; and having an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or more Glucocorticoid-induced tumor necrosis factor receptor (GITR) binding on CD4 + and CD8 + T cells, thereby reversing the inhibitory effects of regulatory T cells (Treg) and co-stimulating and activating effector T cells to exert antitumor effects .
  • GITR Glucocorticoid-induced tumor necrosis factor receptor
  • the anti-VEGF/GITR bispecific antibody of the invention consists of four polypeptide chains that are substantially symmetric about each other, wherein in the two polypeptide chains in the left half and the two polypeptide chains in the right half, Each comprising (i) a single domain antigen binding site; (ii) an antigen binding Fab fragment; wherein said (i) is located at the N-terminus or light chain of said light chain variable domain (VL) of (ii) The C-terminus of the constant region (CL), or the (i) the N-terminus of the heavy chain variable domain (VH) of the (ii) or the C-terminus of the immunoglobulin CH1 domain, and the (i) And (ii) binding to VEGF or GITR, respectively, with or without a linker peptide between (i) and (ii); and (iii) immunoglobulin Fc at the C-terminus of (i) and (ii) Domain.
  • the single domain antigen binding site in the anti-VEGF/GITR bispecific antibody of the invention is a VHH that specifically binds to GITR, and the Fab fragment is an anti-VEGF antibody Fab fragment that specifically binds to VEGF.
  • said VHH that specifically binds to GITR in an anti-VEGF/GITR bispecific antibody of the invention comprises CDR1, SGGGFGD (SEQ ID NO: 26) as shown by GFAGFSS (SEQ ID NO: 25) CDR3 shown in CDR2 and ATDWRKP (SEQ ID NO: 27), or one, two, three, four, five, six with one or more of the three CDRs a sequence of more than one amino acid change (eg, an amino acid substitution or deletion);
  • the anti-VEGF antibody Fab fragment that specifically binds to VEGF in an anti-VEGF/GITR bispecific antibody of the invention comprises a derivative derived from the anti-VEGF antibody Avastin All 6 heavy chain complementarity determining regions (CDRs) and light chain CDRs in the paired heavy chain variable region sequence/light chain variable region sequence set forth in SEQ ID NO: 22/20, or with the 6 CDRs
  • One or more of the CDRs have a sequence of one, two, three, four, five
  • the VHH that specifically binds to GITR in an anti-VEGF/GITR bispecific antibody of the invention comprises, or is substantially derived from, an anti-GITR VHH amino acid sequence set forth in SEQ ID NO: Sequences of the same (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical); of the anti-VEGF/GITR bispecific antibodies of the invention
  • the anti-VEGF antibody Fab fragment that specifically binds to VEGF comprises the paired heavy chain variable region sequence/light chain variable region sequence set forth in SEQ ID NO: 22/20 derived from the anti-VEGF antibody Avastin, or
  • the heavy chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity Sexual sequence.
  • the anti-VEGF/GITR bispecific antibody of the invention consists of four polypeptide chains substantially symmetric about each other, wherein the two polypeptide chains of the left half of the antibody molecule comprise SEQ ID, respectively. a first polypeptide chain represented by NO: 18 and a second polypeptide chain represented by SEQ ID NO: 21; respectively comprising the first polypeptide chain represented by SEQ ID NO: 18 and the SEQ ID NO: 28 a second polypeptide chain; or a sequence substantially identical to any of said sequences (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher)
  • the two polypeptide chains of the right half of the antibody molecule comprise a third polypeptide chain of SEQ ID NO: 18 and a fourth polypeptide chain of SEQ ID NO: 21, respectively; a third polypeptide chain set forth in SEQ ID NO: 18 and a fourth polypeptide chain set forth in SEQ ID NO: 28; or substantially identical to any
  • the invention provides a polynucleotide encoding any one or more polypeptide chains in an antibody molecule of the invention.
  • the invention provides a vector, preferably an expression vector, comprising a polynucleotide encoding any one or more of the polypeptide chains of an antibody molecule of the invention.
  • the invention provides a host cell comprising a polynucleotide or vector of the invention.
  • the host cell is a mammalian cell, preferably a CHO cell, a HEK293 cell; the host cell is a prokaryotic cell, preferably an E. coli cell.
  • the invention provides a method for producing an antibody molecule of the invention, the method comprising the steps of (i) cultivating a host cell of the invention under conditions suitable for expression of the antibody molecule, and (ii) The host cell or the culture medium recovers the antibody molecule.
  • the invention provides an immunoconjugate and a pharmaceutical composition comprising an antibody molecule of the invention.
  • the antibody molecules disclosed herein can be used alone or in combination with other drugs or other therapeutic modalities for the treatment, prevention, and/or diagnosis of diseases such as autoimmune diseases, acute and chronic inflammatory diseases, infectious diseases (eg, chronic infectious diseases or sepsis). ), tumors, etc.
  • the invention provides the use of an antibody molecule, immunoconjugate and pharmaceutical composition of the invention, as a medicament for the treatment and/or prevention of a disease in an individual or as a diagnostic tool for a disease.
  • the individual is a mammal, more preferably a human.
  • the disease is an autoimmune disease, an acute and chronic inflammatory disease, an infectious disease (eg, a chronic infectious disease or sepsis), a tumor.
  • FIGS 1A-1D illustrate four structures of the bispecific antibodies of the invention.
  • 2A-2D show the four structures of the anti-OX40/PD-L1 bispecific antibody Bi-110-112HC, Bi-113- prepared by the present invention by size exclusion chromatography (SEC), respectively. Purity of 112HC, Bi-119-112LC and Bi-122-112LC.
  • Figure 3 shows the binding of anti-OX40/PD-L1 bispecific antibody Bi-119-112LC detected by FACS, and anti-PD-L1 humanized Nb-Fc antibody as a control to CHO cells overexpressing PD-L1.
  • the horizontal axis represents the antibody concentration
  • the vertical axis represents the mean fluorescence intensity (MFI).
  • Figure 4 shows the binding of the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC detected by FACS, and the anti-OX40 antibody ADI-20112 as a positive control to CHO cells overexpressing OX40.
  • the horizontal axis represents the antibody concentration
  • the vertical axis represents the mean fluorescence intensity (MFI).
  • Figure 5 shows the simultaneous binding of anti-OX40/PD-L1 bispecific antibodies to CHO cells overexpressing OX40 and CHO cells overexpressing PD-L1.
  • Figure 6 shows the effect of the anti-OX40/PD-L1 bispecific antibody of the present invention on the binding of human PD-1 to PD-L1, demonstrating that the bispecific antibody Bi-119-112LC of the present invention blocks human PD-1 and The combination of PD-L1.
  • the effect of anti-PD-L1 humanized Nb-Fc and IgG1 as a control was also examined.
  • Figure 7 shows that the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention effectively abolishes the blocking effect of the PD1/PD-L1 interaction on the NFAT signaling pathway, thereby obtaining a fluorescent signal.
  • Figure 8 shows the effect of the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention on the PD-L1-dependent OX40-mediated signaling pathway.
  • the effects of anti-PD-L1 humanized Nb-Fc, ADI-20112, anti-PD-L1 humanized Nb-Fc + ADI-20112 and IgG1 were also examined.
  • FIG. 9 shows the results of Tm values of the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention as determined by differential scanning fluorescence (DSF).
  • Figure 10 shows the activation of human T cells by the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention.
  • the effects of anti-PD-L1 humanized Nb-Fc, ADI-20112 and IgG1 were also examined.
  • FIGS 11A-11B illustrate two structures of the bispecific antibodies of the invention.
  • Figures 12A-12B show the purity of the anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51 prepared by the present invention, respectively, detected by SEC.
  • Figure 13 shows the binding of anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51 detected by FACS to CHO cells overexpressing GITR.
  • the horizontal axis represents the antibody concentration
  • the vertical axis represents the mean fluorescence intensity (MFI).
  • antibody is used herein in its broadest sense to refer to a protein comprising an antigen binding site, encompassing natural and artificial antibodies of various structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), single chain antibodies, intact antibodies, and antibody fragments.
  • full antibody full antibody
  • full length antibody complete antibody
  • intact antibody are used interchangeably herein to refer to a naturally occurring comprising at least two heavy chains (H) interconnected by disulfide bonds. And two light chain (L) glycoproteins.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • the VH and VL regions can be further subdivided into hypervariable regions (which are complementarity determining regions (CDRs) with more conserved regions interposed (framework regions (FR)).
  • CDRs complementarity determining regions
  • FR frame regions
  • Each VH and VL consists of three CDRs and four
  • the FR composition is arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits multiple effector functions.
  • antigen-binding fragment is a portion or portion of an intact or complete antibody that is less than the number of amino acid residues of an intact or fully antibody, which is capable of binding to an antigen or competing with an intact antibody (ie, an intact antibody derived from an antigen-binding fragment). Binding antigen. Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single chain Fv, diabody, single domain antibody (sdAb).
  • the Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains, for example, a Fab fragment can be obtained by digestion of a complete antibody by papain.
  • F(ab') 2 which is a dimer of Fab', is a divalent antibody fragment by digesting a complete antibody under the disulfide bond of the hinge region by pepsin.
  • F(ab') 2 can be reduced under neutral conditions by disrupting the disulfide bond in the hinge region, thereby converting the F(ab') 2 dimer to a Fab' monomer.
  • the Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)).
  • the Fv fragment consists of the VL and VH domains of one arm of the antibody.
  • the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined by a synthetic linker capable of causing the two domains to be produced as a single protein chain using recombinant methods,
  • the VL region and the VH region in a single protein chain are paired to form a single chain Fv.
  • the antibody fragment can be obtained by chemical methods, recombinant DNA methods or protease digestion.
  • single domain antibody or “single variable domain (SVD) antibody” generally refers to an antibody in which a single variable domain (eg, a heavy chain variable domain (VH) or a light chain can be The variable domain (VL), the heavy chain variable domain derived from the camelid heavy chain antibody, and the VH-like single domain (v-NAR) derived from the fish IgNAR confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen.
  • single domain antibodies include single domain antibodies derived from camelids (llamas and camels) and cartilage fish (eg, nurse sharks) (WO 2005/035572).
  • camelized human VH domain means that the transfer of a key element derived from Camelidae VHH to a human VH domain results in the human VH domain no longer needing to be paired with the VL domain to recognize the target antigen, which is camelized.
  • the human VH domain alone confers antigen binding specificity.
  • binding site or "antigen binding site” as used herein denotes a region of an antibody molecule that actually binds to an antigen, including by an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
  • VL antibody light chain variable domain
  • VH antibody heavy chain variable domain
  • a VH/VL pair consisting of a heavy chain variable domain derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) from a shark family IgNAR, a camelized human VH domain, a human A derived camelid antibody heavy chain variable domain.
  • the antibody molecule of the invention comprises at least four antigen binding sites, for example, comprising two single domain antigen binding sites (eg, VHH) and a VH/VL pair in two Fab fragments.
  • the antigen binding site formed.
  • single domain antigen binding site denotes a single variable domain of an antibody molecule (eg, a heavy chain variable domain (VH), a light chain variable domain (VL), derived from a camelid heavy chain antibody Heavy chain variable domain, v-NAR from IgNAR of sharks, camelized human VH domain, humanized camelid antibody heavy chain variable domain, and their recombined single domain ) the area that actually binds to the antigen.
  • the antibody molecule of the invention comprises two single domain antigen binding sites, each of which binds to the same or a different antigen.
  • the antibody molecule of the invention comprises two single domain antigen binding sites, each binding to the same or a different epitope.
  • the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.
  • multispecific antibody refers to an antibody having at least two antigen binding sites, each of the at least two antigen binding sites being different or different from the same epitope of the same antigen. Different epitopes of the antigen bind.
  • the antibodies provided herein are typically multispecific antibodies, such as bispecific antibodies.
  • Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes.
  • a bispecific antibody having binding specificity for a first antigen and a second antigen.
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • an IgG-like immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3) ).
  • VH heavy chain variable region
  • each immunoglobulin light chain has a light chain variable region (VL), also referred to as a light chain variable domain, followed by a light chain constant domain (CL).
  • VL light chain variable region
  • the heavy chain of immunoglobulin can belong to one of five categories, called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG) or ⁇ (IgM), some of which can be further divided into sub- Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG 3 ), ⁇ 4 (IgG 4 ), ⁇ 1 (IgA 1 ), and ⁇ 2 (IgA 2 ).
  • the light chain of an immunoglobulin can be divided into one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.
  • Immunoglobulins consist essentially of two Fab molecules and one Fc domain joined by an immunoglobulin hinge region.
  • Fc domain or "Fc region” is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” comprises two or three constant domains, a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the immunoglobulin Fc domain comprises second and third constant domains (CH2 domain and CH3 domain) derived from two heavy chains of IgG, IgA and IgD class antibodies; or a source comprising The second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of the two heavy chains of the IgM and IgE class antibodies.
  • amino acid residue numbering in the Fc region or heavy chain constant region is according to, for example, Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, The EU numbering system (also known as the EU index) described in 1991 is numbered.
  • effector function refers to those biological activities attributed to the immunoglobulin Fc region that vary with the immunoglobulin isotype.
  • immunoglobulin effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) Cytokine secretion, immune complex-mediated uptake by antigen-presenting cells, down-regulation of cell surface receptors (eg, B cell receptors), and B cell activation.
  • chimeric antibody is an antibody molecule in which (a) changes, replaces or exchanges a constant region or a portion thereof, such that the antigen binding site is different from a different or altered class, effector function and/or species. a region or a completely different molecule (eg, an enzyme, a toxin, a hormone, a growth factor, a drug) that confers new properties to a chimeric antibody; or (b) a variable region or a portion thereof with a different or altered antigen specificity The variable region is changed, replaced or exchanged.
  • a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulin. Due to the replacement into the human constant region, the chimeric antibody retains its specificity in recognizing the antigen while having reduced antigenicity in humans as compared to the original mouse antibody.
  • a “humanized antibody” is an antibody which retains antigen-specific reactivity of a non-human antibody (for example, a mouse monoclonal antibody) and which is less immunogenic when administered to a human as a therapeutic drug. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibody with their human counterpart (ie, the constant region and the portion of the variable region that is not involved in binding is the corresponding portion of the human antibody). See, for example, Padlan, Anatomy of the antibody molecule, Mol. Immun., 1994, 31: 169-217.
  • Other examples of human antibody engineering techniques include, but are not limited to, the Xoma technology disclosed in US 5,766,886.
  • ...valent antibody refers to the number of antigen binding sites present in an antibody molecule.
  • Bivalent, trivalent and tetravalent antibodies refer to the presence of two antigen binding sites, three binding sites and four binding sites, respectively, in the antibody molecule. In one embodiment, the bispecific antibodies reported herein are "tetravalent.”
  • antibody consisting of four polypeptide chains substantially symmetric about left and right means that the antibody molecule consists of four polypeptide chains, including two polypeptide chains to the left of the antibody molecule and two polypeptide chains to the right, and the left side of the antibody molecule 2
  • the sequence of the polypeptide chain and the sequence of the two polypeptide chains on the right have 100% identity or at least 95% or at least 99% identity.
  • flexible linker peptide refers to a linker peptide consisting of amino acids, such as glycine and/or serine residues, used alone or in combination, to link various variable domains in an antibody.
  • the flexible linker peptide is a Gly/Ser linker peptide comprising an amino acid sequence (Gly 4 Ser)n, wherein n is a positive integer equal to or greater than 1, eg, n is a positive integer from 1-7.
  • the flexible linker peptide is (Gly 4 Ser) 2 (SEQ ID NO: 9).
  • binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antigen binding site to bind to a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.
  • affinity or "binding affinity” refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • affinity molecule X for its partner Y can generally dissociation constant (K D) is represented by the solution, the dissociation constant is the ratio of the dissociation rate constant and association rate constant (k dis, respectively and k on) of.
  • K D dissociation constant
  • association rate constant k dis, respectively and k on
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • any macromolecule including substantially all proteins or peptides, can be used as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • the first antigen, the second antigen, and the third antigen are three different antigens.
  • tumor-associated antigen refers interchangeably to a molecule (usually a protein, carbohydrate or lipid) that is expressed completely or as a fragment (eg, MHC/peptide) on the surface of a cancer cell, as compared to normal cells. And the molecule can be used in the preferential targeting of the agent to cancer cells.
  • the tumor associated antigen is a cell surface molecule that is overexpressed in tumor cells as compared to normal cells, eg, 1 fold overexpression, 2 fold overexpression, 3 fold overexpression or more than normal cells Overexpression.
  • the tumor associated antigen is a cell surface molecule that is improperly synthesized in tumor cells, such as a molecule that contains a deletion, addition, or mutation compared to a molecule expressed on a normal cell.
  • the tumor associated antigen is only expressed intact or as a fragment on the cell surface of the tumor cell and is not synthesized or expressed on the surface of normal cells.
  • EGFRvIII epidermal growth factor receptor variant III
  • TAG72 tumor associated glycoprotein 72
  • CEA carcinoembryonic antigen
  • EPCAM epithelial cell adhesion molecule
  • interleukin 11 receptor alpha IL-11Ra
  • VEGFR2 vascular endothelial growth factor receptor 2
  • EGFR epidermal growth factor receptor
  • NCAM neural cell adhesion molecule
  • IGF insulin-like growth factor 1 receptor
  • MAGE-A1 melanoma-associated antigen 1
  • immunocheckpoint means a class of inhibitory signaling molecules present in the immune system that protects against tissue damage by modulating the persistence and strength of immune responses in peripheral tissues and is involved in maintaining tolerance to autoantigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264).
  • Immunological checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, and TIM-3.
  • PD-1 programmed death 1
  • PD-L1 PD-L1
  • PD-L2 PD-L2
  • CTLA-4 cytotoxic T lymphocyte antigen 4
  • LAG-3 LAG-3
  • TIM-3 TIM-3
  • costimulatory molecule refers to a corresponding binding partner on a T cell that specifically binds to a costimulatory ligand to mediate a costimulatory response to T cells, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules that contribute to an effective immune response in addition to antigen receptors or their ligands.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "costimulatory molecule” is OX40, GITR, 4-1BB (ie, CD137), CD27, and/or CD28.
  • cytokine is a generic term for a protein that is released by one cell population and acts as an intercellular medium on another cell.
  • cytokines are lymphokines, mononuclear factors, interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including LIF and kit ligand (KL) and ⁇ -interferon.
  • IL interleukins
  • an “immunoconjugate” is an antibody that is conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.
  • cytotoxic agent refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioisotopes (eg, radioisotopes of At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and Lu); Or drugs (eg, methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, phenylbutyric acid Nitrogen mustard, Zoorubicin or other intercalating agents; growth inhibitors; enzymes and fragments thereof such as lysozyme; antibiotics; small toxins or enzymatically active toxins such as toxins such as bacterial, fungal, plant or animal sources, Included are fragment
  • the “percent identity (%)" of the amino acid sequence means that the candidate sequence is aligned with the specific amino acid sequence shown in the present specification and, if necessary, the vacancy is introduced to achieve the maximum percent sequence identity, and no consideration is given.
  • conservative modifications include substitutions, deletions or additions to polypeptide sequences which result in the replacement of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables that provide functionally similar amino acids are well known in the art.
  • conservatively modified variants are additive relative to the polymorphic variants, interspecies homologs and alleles of the invention and do not exclude them.
  • the following 8 groups contain amino acids that are conservatively substituted: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N) , glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), guanidine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine Acid (C), methionine (M) (see, for example, Creighton, Proteins (1984)).
  • the term "conservative sequence modification” is used to refer to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising an amino acid sequence.
  • N-terminus refers to the last amino acid at the N-terminus
  • C-terminus refers to the last amino acid at the C-terminus
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom.
  • a host cell is any type of cellular system that can be used to produce an antibody molecule of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells, as well as transgenic animals, transgenic plants, or cultured plant tissues or cells within animal tissues.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids incorporated into recombinant polynucleotides, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, glands) Virus and adeno-associated virus).
  • mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, mice and large mouse).
  • domesticated animals eg, cows, sheep, cats, dogs, and horses
  • primates eg, humans and non-human primates such as monkeys
  • rabbits eg, mice and large mouse.
  • rodents eg, mice and large mouse.
  • the individual is a human.
  • treatment refers to the clinical intervention intended to alter the natural course of the disease in an individual being treated. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or mitigating the disease state, and alleviating or improving the prognosis.
  • the antibody molecules of the invention are used to delay the progression of the disease or to slow the progression of the disease.
  • anti-tumor effect refers to a biological effect that can be exhibited by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • tumor and cancer are used interchangeably herein to encompass both solid tumors and liquid tumors.
  • cancer refers to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer.
  • squamous cell carcinoma e.g., epithelial squamous cell carcinoma
  • lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma
  • peritoneal cancer e.g., peritoneal cancer.
  • hepatocellular carcinoma gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, Malignant freckle-like melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as with phagomatoses, edema (such as those associated with brain
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • infectious disease refers to a disease caused by a pathogen, including, for example, a viral infection, a bacterial infection, a parasitic infection, or a fungal infection.
  • the present invention provides a novel antibody molecule that can be used for immunotherapy, prevention, and/or diagnosis of a variety of diseases.
  • the antibody molecule of the invention comprises at least four antigen binding sites which are capable of functioning as monospecific antibodies or multispecific (eg bispecific) antibodies, preferably capable of acting as multispecific (eg bispecific) Antibodies work.
  • the antibody molecule platform constructed herein comprises (i) a single domain antigen binding site; (ii) an antigen binding Fab fragment; wherein said (i) is located in said (ii) light chain variable domain (VL) The C-terminus of the N-terminus or the light chain constant region (CL), or the (i) N-terminus of the heavy chain variable domain (VH) of the (ii) or the C of the immunoglobulin CH1 domain And (i) and (ii) respectively bind the same or different antigens, with or without a linker peptide between (i) and (ii); and at (i) and (ii) (iii) Immunoglobulin Fc domain at the C-terminus.
  • an antibody molecule of the invention has four polypeptide chains comprising two single domain antigen binding sites and two Fab fragments and an Fc region.
  • the single domain antigen binding site of the antibody molecule of the invention and the Fab fragment do not have a linker peptide.
  • a single domain antigen binding site of the antibody molecule of the invention and a Fab fragment have a linker peptide.
  • the type of the linker peptide is not particularly limited.
  • the linker peptide is a peptide having an amino acid sequence of from 1 to 100, in particular from 1 to 50, more particularly from 1 to 20 amino acids in length.
  • the linker peptide is (G 4 S) 2 (SEQ ID NO: 9).
  • a single domain antigen binding site in an antibody molecule of the invention is a single variable domain capable of specifically binding a target antigen epitope with higher affinity, for example, a heavy chain variable domain (VH), a light chain variable structure Domain (VL), heavy chain variable domain derived from camelid heavy chain antibody, v-NAR from IgNAR of sharks, camelized human VH domain, humanized camelid antibody heavy chain variable Domains, and their recombined single domains.
  • the single domain antigen binding site in an antibody molecule of the invention is a heavy chain variable domain derived from a camelid heavy chain antibody, a camelized human VH domain, and/or a humanized camel Family antibody heavy chain variable domain.
  • camelid species such as camelids, alpaca, dromedary, llama and guanaco have been characterized in the prior art.
  • a heavy chain variable domain of a camelid heavy chain antibody having high affinity for a target antigen (this region is also referred to as VHH) can be obtained by a genetic engineering method. See U.S. Patent No. 5,759,808, issued June 2, 1998.
  • the amino acid sequence of Camelidae VHH can be recombinantly altered to obtain sequences that more realistically mimic human sequences, i.e., "humanized,” thereby reducing the antigenicity of Camelidae VHH to humans.
  • key elements derived from Camelidae VHH can be transferred to the human VH domain to obtain a camelized human VH domain.
  • the single domain antigen binding site in the antibody molecule of the invention is a humanized VHH directed against PD-L1 having the sequence set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2. Amino acid sequence.
  • the single domain antigen binding site in the antibody molecule of the invention is a VHH directed against GITR having the amino acid sequence set forth in SEQ ID NO:24.
  • the molecular weight of VHH is one tenth of the molecular weight of a human IgG molecule and has a physical diameter of only a few nanometers.
  • VHH itself has extremely high thermal stability, is stable to extreme pH and proteolytic digestion, and has low antigenicity. Therefore, in one embodiment of the antibody molecule of the present invention, VHH is included as a building block for stability of the antibody molecule of the present invention, The low antigenicity of human subjects contributes.
  • the Fab fragments in the antibody molecules of the invention are capable of specifically binding to a target antigen epitope with higher affinity.
  • the Fab fragment is a Fab fragment of an immunoglobulin comprising a peptide consisting of an immunoglobulin light chain variable region (VL) and an immunoglobulin light chain constant region (CL); a peptide consisting of an immunoglobulin heavy chain variable region (VH) and an immunoglobulin heavy chain constant region 1 (CH1); wherein the CL region and the CH1 region are optionally covalently linked by disulfide bonds and heterodimerized Fab fragments.
  • VL immunoglobulin light chain variable region
  • CL immunoglobulin light chain constant region
  • CH1 immunoglobulin heavy chain constant region 1
  • the Fab fragment is a light chain variable region (VL) and a light chain variable region (VL) exchanged Fab fragment of an immunoglobulin Fab fragment, comprising a variable immunoglobulin light chain a peptide consisting of a region (VL) and a heavy chain constant region (CH1); and comprising a peptide consisting of an immunoglobulin heavy chain variable region (VH) and a light chain constant region (CL); wherein the CL region and the CH1 region
  • the Fab fragment is heterodimericly heterologously covalently linked by a disulfide bond.
  • the Fab fragment is a light chain constant region (CL) and a heavy chain constant region (CH1) exchanged Fab fragment of an immunoglobulin Fab fragment comprising an immunoglobulin heavy chain variable region a peptide consisting of (VH) and a light chain constant region (CL); and comprising a peptide consisting of an immunoglobulin light chain variable region (VL) and a heavy chain constant region (CH1); wherein the CL region and the CH1 region are The Fab fragment is heterodimericly heterologously covalently linked by a disulfide bond.
  • Fab fragment in an antibody molecule of the invention does not comprise a disulfide bond.
  • the two strands of the Fab fragment can be engineered in such a way as to stably interact without the need for a disulfide bond.
  • both strands of a Fab fragment can be engineered to remove a cysteine residue, and the two strands of the Fab fragment still stably interact and function as a Fab.
  • the two strands of the Fab fragment are mutated to promote a stable interaction between the two strands.
  • a genetic modification strategy can be used (see, for example, John BBRidgway et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, 1996.9(7): p.
  • a Fab fragment in an antibody molecule of the invention is derived from a monoclonal antibody and can be derived from any type of antibody, including IgA, IgM, IgD, IgG, IgE, and subtypes thereof, eg, IgGl, IgG2, IgG3, and IgG4.
  • the light chain domain can be derived from a kappa chain or a lambda chain.
  • the Fab fragments used herein can also be prepared by recombinant.
  • the CH1 domain, CL domain in a Fab fragment of an antibody molecule of the invention is derived from or substantially identical to a corresponding portion of a human immunoglobulin (eg, at least 80%, 85%, 90%) , 92%, 95%, 97%, 98%, 99% or more of the same sequence.
  • the immunoglobulin Fc domain in the antibody molecule of the invention is capable of extending the in vivo half-life of the antibody of the invention and providing effector function. See, for example, International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375.
  • the hinge region having a "CPPC" amino acid residue is contained in the Fc domain of the second polypeptide chain and the fourth polypeptide chain of the antibody molecule of the invention, respectively, and/or comprises Y349C and S354C, respectively. (According to Kabat's "EU No.”), whereby the second polypeptide chain and the fourth polypeptide chain of the antibody molecule of the present invention form an interchain disulfide bond in the Fc region, which also contributes to the antibody molecule of the present invention. Correct pairing of the two polypeptide chains and the fourth polypeptide chain.
  • the immunoglobulin Fc domain of an antibody molecule of the invention also employs a "binding" technique that modifies the interface between different strands of an antibody molecule of the invention to facilitate the antibody molecule of the invention
  • a "binding" technique that modifies the interface between different strands of an antibody molecule of the invention to facilitate the antibody molecule of the invention
  • This technique involves introducing a "bump" at the interface of one strand, introducing a corresponding "hole” at the interface of the other strand to be paired with, such that the projection can be placed in the void.
  • the first preferred interface comprises the CH3 domain of the heavy chain constant domain of one strand and the CH3 domain of the heavy chain constant domain of the other strand to be paired with.
  • the bulges can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one strand with a larger side chain (eg, tyrosine or tryptophan).
  • a larger side chain eg, tyrosine or tryptophan
  • the interface of the CH3 domain of the heavy chain constant domain of another strand to be paired is identical or similar to the bulge Compensatory holes of size.
  • a second preferred interface is the CL domain comprising the light chain and the CH1 domain of the heavy chain of the Fab fragment described above, which facilitates the correct formation between the two strands of the Fab fragment by constructing a bulge-hole interaction. Heterodimerization.
  • the Fc region of an antibody molecule of the invention comprises a modification to the binding affinity of an Fc receptor.
  • the Fc receptor is an Fc gamma receptor, in particular a human Fc gamma receptor.
  • the Fc receptor is an activating Fc receptor.
  • the modification reduces the effector function of an antibody molecule of the invention.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the modification is in the Fc region of the immunoglobulin molecule, particularly in its CH2 region.
  • the immunoglobulin molecule comprises an amino acid substitution at position 329 (EU numbering) of the immunoglobulin heavy chain.
  • an antibody molecule of the invention comprises an amino acid substitution at positions 234 and 235 (EU numbering) of the immunoglobulin heavy chain.
  • the amino acid substitutions are L234A and L235A (LALA mutations) (Armour KL et al, Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol, 1999. 29(8): 2613 twenty four).
  • an antibody molecule of the invention comprises an amino acid substitution at positions 234, 235 and 329 of the immunoglobulin heavy chain (EU numbering).
  • the immunoglobulin molecule comprises amino acid substitutions L234A, L235A and P329G (EU numbering) in the immunoglobulin heavy chain.
  • At least one single domain antigen binding site eg, two single domain antigen binding sites
  • at least one Fab fragment in an antibody molecule of the invention are capable of specifically binding at least one antigen.
  • the antibody molecule of the present invention binds two or more antigens, whereby the antibody molecule of the present invention is a multispecific antibody molecule, for example, a bispecific antibody molecule.
  • antigens include, but are not limited to, cytokines, growth factors, hormones, signaling proteins, inflammatory mediators, ligands, cell surface receptors or fragments thereof.
  • an antibody molecule of the invention inhibits signaling pathways of a plurality (eg, two) of immunological checkpoint molecules, eg, the antibody molecule of the invention has a first binding specificity for PD-L1 and for TIM -3.
  • the antibody molecule of the invention inhibits signaling pathways of immunological checkpoint molecules and signaling pathways of agonistic costimulatory molecules, eg, the antibody molecules of the invention are directed against PD-L1, TIM-3, LAG- 3.
  • the signaling pathway functions by signaling the signaling pathway of the costimulatory molecule.
  • the antibody molecules of the invention inhibit signaling pathways of immunological checkpoint molecules and inhibit abnormal angiogenesis
  • the antibody molecules of the invention are directed against PD-L1, TIM-3, LAG-3, PD- 1 or a bispecific antibody molecule of PD-L2 with a first binding specificity and a second binding specificity for VEGF or VEGF receptor, by inhibiting the signaling pathway of the immunological checkpoint molecule and by inhibiting VEGF, VEGF The signal transduction pathway of the body functions.
  • the antibody molecules of the invention agonize signaling pathways of multiple (eg, two) costimulatory molecules, eg, the antibody molecules of the invention have a first binding specificity for OX40 and for GITR, 4- A second binding specific bispecific antibody molecule of 1BB, CD27 or CD28 acts by agonizing the signaling pathway of the costimulatory molecule.
  • an antibody molecule of the invention agonizes a signaling pathway of a costimulatory molecule and inhibits abnormal angiogenesis
  • the antibody molecule of the invention has a first binding to OX40, GITR, 4-1BB, CD27 or CD28 Bispecific antibody molecules that are specific and specific for the second binding specificity of VEGF or VEGF receptors act by agonizing the signaling pathway of the costimulatory molecule and by inhibiting the signaling pathways of VEGF, VEGF receptors.
  • the antibody molecule of the invention inhibits abnormal angiogenesis, inhibits signaling pathways of immunological checkpoint molecules, and signaling pathways of agonistic costimulatory molecules, eg, the antibody molecules of the invention are directed against VEGF or VEGF receptors First binding specificity, second binding specificity for PD-L1, TIM-3, LAG-3, PD-1 or PD-L2 and third binding to OX40, GITR, 4-1BB, CD27 or CD28
  • a specific trispecific antibody molecule functions by inhibiting a signaling pathway of VEGF, a VEGF receptor, a signal transduction pathway that inhibits the immunological checkpoint molecule, and a signaling pathway that agonizes the costimulatory molecule.
  • an antibody molecule of the invention has any of the structures illustrated in Figures 1A-1D of the specification.
  • an exemplary antibody molecule of the invention is a four-chain antibody molecule comprising two Fab fragments, a single domain antigen binding site located at the C-terminus of the light chain constant region (CL) of each Fab fragment, respectively. And an immunoglobulin Fc domain which is the C-terminus of the antibody molecule of the present invention, wherein there is or does not have a linker peptide between the C-terminus of the light chain constant region (CL) of the Fab fragment and the single domain antigen binding site.
  • an exemplary antibody molecule of the invention is a four-chain antibody molecule comprising two Fab fragments, a single domain antigen binding at the N-terminus of the light chain variable domain (VL) of each Fab fragment, respectively.
  • an exemplary antibody molecule of the invention is a four-chain antibody molecule comprising two Fab fragments, a single domain antigen binding site located at the C-terminus of the CH1 domain of each Fab fragment, and as a native An immunoglobulin Fc domain of the C-terminus of an antibody molecule, wherein there is or no linker peptide between the C-terminus of the Fab fragment CH1 domain and the single domain antigen binding site.
  • an exemplary antibody molecule of the invention is a four-chain antibody molecule comprising two Fab fragments, single domain antigen binding at the N-terminus of the heavy chain variable domain (VH) of each Fab fragment, respectively.
  • the antibody molecule of the invention is an anti-OX40/PD-L1 bispecific antibody or a multispecific antibody.
  • OX40 (also known as CD134, TNFRSF4, and ACT35) is a member of the cell surface glycoprotein and tumor necrosis factor (TNF) receptor superfamily, expresses on T lymphocytes and provides a costimulatory signal for the proliferation and survival of activated T cells.
  • TNF tumor necrosis factor
  • OX40 was originally described as a T cell activation marker on rat CD4 T cells (Paterson DJ et al, Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mol Immunol. 1987; 24:1281 -1290) and subsequently shown to be up-regulated in TCR recruitment (Mallett S.
  • OX40 signaling promotes costimulatory signals to T cells, resulting in enhanced cell proliferation, survival, effector function, and migration (Gramaglia I et al., Ox-40ligand: a potent costimulatory molecule for sustaining primary CD4T cell responses. J Immunol. 1998; 161:6510–6517; Gramaglia I et al, The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion. J Immunol. 2000; 165:3043–3050).
  • Anti-OX40 antibodies that are OX40 agonists are disclosed in the prior art.
  • the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-OX40 antibody mAb 106-222 and humanized 106-222 (Hu106) are disclosed in WO 2012/027328; anti-OX40 antibody mAb 119-122 and The amino acid sequences of the heavy chain variable region and the light chain variable region of humanized 119-122 (Hu119).
  • Anti-OX40 antibodies that are OX40 agonists are also disclosed in U.S. Patent No. 7,959,925, PCT Publication No. WO 2006/121810, and Chinese Patent Application No. 201710185399.9.
  • the anti-OX40 antibody is capable of activating OX40, thereby inducing proliferation of effector T lymphocytes and promoting an immune response against tumor cells expressing tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • PD-L1 (also known as differentiation antigen cluster 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40 kDa type I transmembrane protein. PD-L1 binds to its receptor PD-1 present on activated T cells, downregulating T cell activation (Latchman et al, 2001 Nat Immunol 2: 261-8; Carter et al, 2002 Eur J Immunol 32: 634-43 ). PD-L1 expression has been found in many cancers, including human lung cancer, ovarian cancer, colon cancer, and various myeloma, and PD-L1 expression is often associated with poor prognosis of cancer (Iwai et al.
  • the anti-OX40/PD-L1 bispecific antibody or multispecific antibody of the invention simultaneously targets at least OX40 and PD-L1 at the same time, and the Fab fragment and the single domain antigen binding site respectively bind to the OX40 or PD-L1 molecule, and can block
  • the inhibitory PD-1/PD-L1 signaling pathway is disrupted and the OX40/OX40 ligand signaling pathway in T cells and natural killer (NK) cells is activated to promote an immune response against the disease.
  • an antibody molecule of the invention comprises a single domain antigen binding site that specifically binds to PD-L1 and a Fab fragment that specifically binds OX40. In one embodiment, an antibody molecule of the invention comprises a single domain antigen binding site that specifically binds OX40 and a Fab fragment that specifically binds to PD-L1.
  • the Fab fragment that specifically binds to PD-L1 or OX40 it comprises an anti-PD-L1 antibody (for example, the anti-PD-L1 antibody exemplified above) which is derived from any of the prior art and which is developed in the future.
  • an anti-PD-L1 antibody for example, the anti-PD-L1 antibody exemplified above
  • an anti-PD-L1 antibody for example, the anti-PD-L1 antibody exemplified above
  • the anti-OX40 antibody is ADI-20112 having the heavy chain amino acid sequence set forth in SEQ ID NO: 10 and the light chain amino acid sequence set forth in SEQ ID NO: 15.
  • the single domain antigen binding site that specifically binds to PD-L1 or OX40, it comprises a heavy chain variable domain (VH), a light chain variable domain (VL) that specifically binds to PD-L1 or OX40 a heavy chain variable domain in camelid antibodies consisting of only two heavy chains, which are naturally free of light chains from camelid serum, a VH-like single domain of IgNAR from sharks, and a camelized human VH structure. Domain, humanized camelid antibody heavy chain variable domain.
  • an anti-OX40/PD-L1 bispecific antibody of the invention comprises two Fab fragments that specifically bind OX40 and two single domain antigen binding sites that specifically bind to PD-L1 (eg, VHH) ) has any of the structures illustrated in Figures 1A-1D, respectively.
  • the two Fab fragments that specifically bind to OX40 specifically bind to the same epitope or different epitopes on the OX40 molecule; the two single domain antigen binding sites that specifically bind to PD-L1 specifically bind to PD-L1 The same epitope or different epitopes on the molecule.
  • the Fab fragment that specifically binds OX40 in an anti-OX40/PD-L1 bispecific antibody of the invention comprises the paired weight of SEQ ID NO: 11/7 derived from the anti-OX40 antibody ADI-20112 All 6 heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the chain variable region sequence/light chain variable region sequence, or one or two with one or more of the 6 CDRs Sequences of one, three, four, five, six or more amino acid changes (eg, amino acid substitutions or deletions).
  • CDRs heavy chain complementarity determining regions
  • the Fab fragment that specifically binds OX40 in an anti-OX40/PD-L1 bispecific antibody of the invention comprises the paired weight of SEQ ID NO: 11/7 derived from the anti-OX40 antibody ADI-20112 a chain variable region sequence/light chain variable region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95 with the paired heavy chain variable region sequence/light chain variable region sequence %, 96%, 97%, 98%, 99% or more sequences of sequence identity.
  • the single domain antigen binding site that specifically binds to PD-L1 in an anti-OX40/PD-L1 bispecific antibody of the invention comprises CDR1, SEQ ID set forth in SEQ ID NO: CDR2 represented by NO: 4 and CDR3 represented by SEQ ID NO: 5, or one, two, three, four, five, six or one or more of the three CDRs More sequences of amino acid changes (eg, amino acid substitutions or deletions).
  • the single domain antigen binding site that specifically binds to PD-L1 in an anti-OX40/PD-L1 bispecific antibody of the invention comprises SEQ ID NO: 1 and/or SEQ ID NO The amino acid sequence shown in 2, or a sequence substantially identical thereto (for example, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).
  • the type of the heavy chain constant region CH1 domain and the Fc region (including the CH2 domain, the CH3 domain, and the optional CH4 domain) in the anti-OX40/PD-L1 bispecific antibody of the present invention is not particularly limited, and is preferably
  • the corresponding domain derived from the IgG1, IgG2 or IgG4 immunoglobulin heavy chain constant region is substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more of the same sequence.
  • the heavy chain constant region CH1 domain and Fc region are derived from, or substantially identical to, the CH1 domain and the Fc region of the heavy chain constant region of a human IgG1 immunoglobulin (eg, at least 80%, 85) Sequence of %, 90%, 92%, 95%, 97%, 98%, 99% or more of the same).
  • an anti-OX40/PD-L1 bispecific antibody of the invention comprises a CH1 domain and an Fc region of an IgG4 (eg, human IgG4) heavy chain constant region.
  • an anti-OX40/PD-L1 bispecific antibody of the invention comprises a CH1 domain and an Fc region of an IgG1 (eg, human IgG1) heavy chain constant region.
  • an anti-OX40/PD-L1 bispecific antibody of the invention comprises a CH1 domain of an IgG4 (eg, human IgG4) heavy chain constant region and an Fc of an IgG1 (eg, human IgG1) heavy chain constant region a region; or a Fc region comprising a IgG1 (eg, human IgG1) heavy chain constant region and an IgG4 (eg, human IgG4) heavy chain constant region.
  • the Fc domain of the second polypeptide chain and the fourth polypeptide chain of the anti-OX40/PD-L1 bispecific antibody of the invention comprises a hinge region having a "CPPC" amino acid residue, respectively, and / Or separately comprising Y349C and S354C (according to Kabat's "EU numbering"), whereby the second polypeptide chain and the fourth polypeptide chain of the anti-OX40/PD-L1 bispecific antibody of the invention form an interchain disulfide bond in the Fc region. Thereby, the correct pairing of the second polypeptide chain and the fourth polypeptide chain is stabilized.
  • the second polypeptide chain and/or the fourth polypeptide chain of an anti-OX40/PD-L1 bispecific antibody of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function.
  • the amino acid substitution is a LALA mutation.
  • the anti-OX40/PD-L1 bispecific antibody of the invention comprises a kappa light chain constant region and/or a lambda light chain constant region, eg, a human kappa light chain constant region and/or a human lambda light chain is constant Area.
  • the light chain constant region comprises or is substantially identical to the amino acid sequence set forth in SEQ ID NO: 8 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) , 98%, 99% or more of the same sequence.
  • the second polypeptide chain and the fourth polypeptide chain of the anti-OX40/PD-L1 bispecific antibody of the invention comprise a stable association of "binding" in the respective Fc domains, respectively.
  • an amino acid substitution T366W is included in one of the second polypeptide chain and the fourth polypeptide chain, and is included in the other of the second polypeptide chain and the fourth polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering).
  • the immunoglobulin CHI1 domain and the CL domain of the anti-OX40/PD-L1 bispecific antibody of the invention comprise a bulge or a hole, respectively, and the bulge or empty in the CH1 domain
  • the wells can be placed in the holes or bulges in the CL domain, respectively, such that the first polypeptide chain and the second polypeptide chain of the anti-OX40/PD-L1 bispecific antibody of the invention also form an "inclusion" with each other.
  • the stable association of the buckle is described in one embodiment, the immunoglobulin CHI1 domain and the CL domain of the anti-OX40/PD-L1 bispecific antibody of the invention.
  • the anti-OX40/PD-L1 bispecific antibody of the invention consists of four polypeptide chains that are substantially symmetric about each other, wherein the two polypeptide chains of the left half of the antibody molecule comprise SEQ ID, respectively.
  • the anti-OX40/PD-L1 bispecific antibody of the present invention is capable of binding to both PD-L1 and OX40 proteins simultaneously, and maintains the affinity constant of the parent antibody, thereby being able to block the PD-1/PD-L1 signaling pathway and Activate the OX40/OX40 ligand signaling pathway in T cells and natural killer (NK) cells.
  • the anti-OX40/PD-L1 bispecific antibodies of the invention can be used in the treatment, prevention or diagnosis of diseases associated with such signaling pathways.
  • the antibody molecule of the invention is an anti-VEGF/GITR bispecific antibody or a multispecific antibody.
  • VEGF Vascular endothelial growth factor
  • VPF vascular permeability factor
  • Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid, Science, 1983, 219 (4587): 983-985
  • VEGF is a signaling protein produced by cells that stimulate blood vessels.
  • VEGF is a subfamily of growth factors that are a major class of signaling proteins involved in angiogenesis.
  • Vascular endothelial growth factor and vascular endothelial growth inhibitory factor are simultaneously present in normal tissues and remain relatively balanced, which allows human blood vessels to be normally produced and differentiated.
  • VEGF family molecules are proliferating, and the imbalance between regulation and angiogenesis inhibitors is greatly promoted, thereby greatly promoting the proliferation and migration of vascular endothelial cells, and improving vascular permeability.
  • Inhibition of tumor cell apoptosis provides a good microenvironment for tumor growth and metastasis (Lapeyre-Prost A et al, Immunomodulatory Activity of VEGF in Cancer, Int Rev Cell Mol Biol. 2017; 330:295-342).
  • the VEGF family contains six closely related polypeptides, each of which is a highly conserved homodimeric glycoprotein, with six subtypes: VEGF-A, -B, -C, -D, -E, and placental growth factor ( Placental growth factor (PLGF)), with molecular weights ranging from 35 to 44 kDa.
  • VEGF-A including its splices such as VEGF 165
  • VEGF-A is associated with microvessel density in some solid tumors
  • the concentration of VEGF-A in tissues is associated with the prognosis of solid tumors such as breast, lung, prostate and colon cancers. .
  • each VEGF family member is mediated by one or more of the cell surface VEGF receptor (VEGFR) family, including VEGFR1 (also known as Flt-1), VEGFR2 (also known as KDR). Flk-1), VEGFR3 (also known as Flt-4), etc., wherein VEGFR1 and VEGFR2 are closely related to angiogenesis, and VEGF-C/D/VEGFR3 is closely related to lymphangiogenesis.
  • VEGFR1 also known as Flt-1
  • VEGFR2 also known as KDR
  • Flk-1 Flk-1
  • VEGFR3 also known as Flt-4
  • Bevacizumab (trade name: Avastin), developed by Genentech, is a recombinant human-mouse chimeric anti-VEGF antibody that blocks VEGFR from inactivation by blocking the binding of VEGF-A to VEGFR. This exerts an anti-angiogenic effect.
  • Bevacizumab is currently used for first-line treatment of metastatic colorectal cancer, and may be used for the treatment of metastatic lung cancer, breast cancer, pancreatic cancer, and kidney cancer in the future. Bevacizumab is also one of the more successful antibody drugs developed.
  • Glucocorticoid-induced tumor necrosis factor receptor (GITR, also known as TNFRSF18, activation-inducible TNFR family members (AITR), CD357 and GITR-D), is a tumor necrosis factor (tumor necrosis factor) , the 18th member of the TNF) receptor superfamily. Initially identified in a murine T cell line treated with dexamethasone (Nocentini G et al, A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis, Proc Natl Acad Sci U S A. 1997; 94(12): 6216-21).
  • TNF receptor superfamily include CD40, CD27, 4-1BB, and OX40.
  • GITR expression is lower in naive CD4+ and CD8+ cells, it is constitutively expressed in regulatory T cells (Tone M et al., Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells, Proc Natl Acad Sci U S A. 2003; 100 (25): 15059-64).
  • GITR expression is induced on effector T cells, effector T cell activation, proliferation, and cytokine production are promoted.
  • CD4+CD25+ regulatory T cells Shimizu used mixed culture repression assays to report that GITR activation inhibits Treg function (Shimizu J et al., Stimulation of CD25(+)CD4(+) regulatory T cells through GITR Breaks immunological self-tolerance, Nature Immunology 2002; 3: 135-42).
  • Anti-GITR antibody DTA-1 mediated GITR stimulation promotes anti-tumor immunity in a variety of tumor models (Cohen AD, Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor Accumulation, PLoS One. 2010; 5(5): e10436; Coe D et al, Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy, Cancer Immunol Immunother, 2010; 59(9): 1367- 77).
  • GITR is activated by binding to GITR ligand (GITRL), which is mainly expressed on APC. After activation, GITR can increase resistance to tumor and viral infection, participate in autoimmune processes/inflammatory processes, and regulate leukocyte extravasation.
  • GITRL GITR ligand
  • Anti-GITR antibodies are described in U.S. Patent No. 7,025,962, European Patent No. 1,947,183 B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent No. EP 1866339, PCT Publication No. WO 2011/028683, U.S. Patent No. 8,709,424, PCT Publication No. WO 2013/039954, International Publication No. WO 2013/039954, US Publication No. US 2014/0072566, International Publication No. WO 2015/026684, PCT Publication No. WO 2005/007190, PCT Publication No. WO 2007/133822 PCT Publication No.
  • the anti-VEGF/GITR bispecific antibody or multispecific antibody of the present invention targets at least VEGF and GITR at the same time, and the Fab fragment and the single domain antigen binding site respectively bind to the VEGF or GITR molecule, and can block the VEGF family signaling pathway. And the function of activating effector T cells and suppressing Treg.
  • an antibody molecule of the invention comprises a single domain antigen binding site that specifically binds to GITR and a Fab fragment that specifically binds to VEGF. In one embodiment, an antibody molecule of the invention comprises a single domain antigen binding site that specifically binds to VEGF and a Fab fragment that specifically binds to GITR.
  • the Fab fragment that specifically binds GITR or VEGF comprises an anti-GITR antibody (e.g., an anti-GITR antibody exemplified above) derived from any of the prior art and an anti-GITR antibody VH/VL developed in the future.
  • an anti-GITR antibody e.g., an anti-GITR antibody exemplified above
  • the anti-VEGF antibody is Avastin having the heavy chain amino acid sequence set forth in SEQ ID NO: 19 and the light chain amino acid sequence set forth in SEQ ID NO: 18.
  • the single domain antigen binding site that specifically binds GITR or VEGF, it comprises a heavy chain variable domain (VH), a light chain variable domain (VL), which is specifically binding to GITR or VEGF, from Camelidae
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the heavy chain variable domain of a camel antibody consisting of only two heavy chains, which is naturally free of light chains, the VH-like single domain of IgNAR from sharks, the camelized human VH domain, human origin Camelid antibody heavy chain variable domain.
  • an anti-VEGF/GITR bispecific antibody of the invention comprises two Fab fragments that specifically bind to VEGF and two single domain antigen binding sites (eg, VHH) that specifically bind to GITR, each having Any of the structures illustrated in FIGS. 1A, 1B, 1D, 11A, and 11B.
  • the two Fab fragments that specifically bind to VEGF specifically bind to the same epitope or different epitopes on the VEGF molecule; the two single domain antigen binding sites that specifically bind to GITR specifically bind to the same on the GITR molecule Epitope or different epitopes.
  • the Fab fragment that specifically binds to VEGF in an anti-VEGF/GITR bispecific antibody of the invention comprises a pair of heavy chain variable regions of SEQ ID NO: 22/20 derived from the anti-VEGF antibody Avastin All six heavy chain complementarity determining regions (CDRs) and light chain CDRs contained in the sequence/light chain variable region sequence, or one, two, three with one or more of the six CDRs Sequence of four, five, six or more amino acid changes (eg, amino acid substitutions or deletions).
  • CDRs heavy chain complementarity determining regions
  • the Fab fragment that specifically binds to VEGF in an anti-VEGF/GITR bispecific antibody of the invention comprises a pair of heavy chain variable regions of SEQ ID NO: 22/20 derived from the anti-VEGF antibody Avastin a sequence/light chain variable region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with the paired heavy chain variable region sequence/light chain variable region sequence , 97%, 98%, 99% or more sequences of sequence identity.
  • the single domain antigen binding site that specifically binds to GITR in an anti-VEGF/GITR bispecific antibody of the invention comprises CDR1, SGGGFGD (SEQ ID NO: 25) ID NO: 26) CDR3 shown by CDR2 and ATDWRKP (SEQ ID NO: 27), or one, two, three, four, five with one or more of the three CDRs Sequence of one, six or more amino acid changes (eg, amino acid substitutions or deletions).
  • the single domain antigen binding site that specifically binds to GITR in an anti-VEGF/GITR bispecific antibody of the invention comprises, or is essential to, the amino acid sequence set forth in SEQ ID NO: Sequences that are identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).
  • the type of the heavy chain constant region CH1 domain and the Fc region (including the CH2 domain, the CH3 domain, and the optional CH4 domain) in the anti-VEGF/GITR bispecific antibody of the present invention is not particularly limited, and is preferably derived from
  • the corresponding domain of the IgG1, IgG2 or IgG4 immunoglobulin heavy chain constant region is substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%) Or more of the same sequence.
  • the heavy chain constant region CH1 domain and Fc region are derived from or substantially identical to the CH1 domain and the Fc region of the heavy chain constant region of a human IgG1 immunoglobulin (eg, at least 80%, 85) Sequence of %, 90%, 92%, 95%, 97%, 98%, 99% or more of the same).
  • an anti-VEGF/GITR bispecific antibody of the invention comprises a CH1 domain and an Fc region of an IgG4 (eg, human IgG4) heavy chain constant region. In one embodiment, an anti-VEGF/GITR bispecific antibody of the invention comprises a CH1 domain and an Fc region of an IgG1 (eg, human IgG1) heavy chain constant region.
  • an anti-VEGF/GITR bispecific antibody of the invention comprises a CH1 domain of an IgG4 (eg, human IgG4) heavy chain constant region and an Fc region of an IgG1 (eg, human IgG1) heavy chain constant region;
  • the Fc region comprising the IgG1 (eg, human IgG1) heavy chain constant region and the IgG4 (eg, human IgG4) heavy chain constant region.
  • the Fc domains of the second polypeptide chain and the fourth polypeptide chain of the anti-VEGF/GITR bispecific antibody of the invention comprise a hinge region having a "CPPC" amino acid residue, respectively, and/or respectively Containing Y349C and S354C ("EU number" according to Kabat), whereby the second polypeptide chain and the fourth polypeptide chain of the anti-VEGF/GITR bispecific antibody of the invention form an interchain disulfide bond in the Fc region, Thus, the correct pairing of the second polypeptide chain and the fourth polypeptide chain is stabilized.
  • the second polypeptide chain and/or the fourth polypeptide chain of an anti-VEGF/GITR bispecific antibody of the invention comprises an amino acid mutation in the Fc domain that affects antibody effector function.
  • the amino acid substitution is a LALA mutation.
  • an anti-VEGF/GITR bispecific antibody of the invention comprises a kappa light chain constant region and/or a lambda light chain constant region, eg, a human kappa light chain constant region and/or a human lambda light chain constant region.
  • the light chain constant region comprises or is substantially identical to the amino acid sequence set forth in SEQ ID NO: 8 (eg, at least 80%, 85%, 90%, 92%, 95%, 97%) , 98%, 99% or more of the same sequence.
  • the second polypeptide chain and the fourth polypeptide chain of the anti-VEGF/GITR bispecific antibody of the invention comprise a stable association of "binding" in the respective Fc domains, respectively.
  • an amino acid substitution T366W is included in one of the second polypeptide chain and the fourth polypeptide chain, and is included in the other of the second polypeptide chain and the fourth polypeptide chain Amino acid substitutions T366S, L368A and Y407V (EU numbering).
  • the immunoglobulin CHI1 domain and the CL domain of the anti-VEGF/GITR bispecific antibody of the invention comprise a bulge or a hole, respectively, and the bulge or cavity in the CH1 domain may Separately placed in the holes or bulges in the CL domain, such that the first polypeptide chain and the second polypeptide chain of the anti-VEGF/GITR bispecific antibody of the invention also form a "binding" stability Association.
  • the anti-VEGF/GITR bispecific antibody of the invention consists of four polypeptide chains that are substantially symmetric about each other, wherein the two polypeptide chains of the left half of the antibody molecule comprise SEQ ID NO: a first polypeptide chain represented by 18 and a second polypeptide chain of SEQ ID NO: 21; comprising a first polypeptide chain of SEQ ID NO: 18 and a second of SEQ ID NO: 28, respectively a polypeptide chain; or a sequence substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher) to any of said sequences; Wherein the two polypeptide chains of the right half of the antibody molecule comprise a third polypeptide chain of SEQ ID NO: 18 and a fourth polypeptide chain of SEQ ID NO: 21, respectively; a third polypeptide chain represented by NO: 18 and a fourth polypeptide chain represented by SEQ ID NO: 28; or substantially identical to any of the sequences (eg.,
  • the anti-VEGF/GITR bispecific antibody of the present invention is capable of binding to both GITR and VEGF proteins, and maintains the affinity constant of the parent antibody, thereby blocking the VEGF family signaling pathway and activating effector T cells and natural killing (NK) GITR/GITR ligand signaling pathway in cells.
  • the anti-VEGF/GITR bispecific antibodies of the invention can be used in the treatment, prevention or diagnosis of diseases associated with such signaling pathways.
  • amino acid sequence variants of the bispecific antibodies exemplified herein are contemplated.
  • Amino acid sequence variants of bispecific antibodies can be made by introducing appropriate modifications to the nucleotide sequence encoding the bispecific antibody or by peptide synthesis. Such modifications include, for example, deletion of residues from within the amino acid sequence of the antibody and/or insertion of residues into and/or substitution of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses a desired characteristic, such as antigen binding.
  • Amino acids can be grouped according to common side chain properties:
  • Non-conservative substitutions will cause members of one of these categories to be exchanged for members of another classification.
  • the antibody molecules of the invention are capable of recombinant fusion or chemical conjugation (including covalent and non-covalent conjugation) to a heterologous protein or polypeptide to produce a fusion protein.
  • Methods of fusing or conjugating a protein, polypeptide or peptide to an antibody are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929 and EP 367,166.
  • antibody molecules of the invention can be fused to a labeling sequence (e.g., a peptide) to facilitate purification.
  • the labeled amino acid sequence is a hexahistidine peptide, such as the one provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), etc., many of which are commercially available. of. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine provides convenient purification of the fusion protein.
  • peptide tags for purification include, but are not limited to, hemagglutinin ("HA") tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell 37: 767) and "flag" label.
  • HA hemagglutinin
  • an antibody molecule of the invention is conjugated to a diagnostic or detectable agent.
  • a diagnostic or detectable agent e.g., to determine the efficacy of a particular therapy
  • Such antibodies can be used as part of a clinical test (eg, to determine the efficacy of a particular therapy) for monitoring or predicting the onset, formation, progression, and/or severity of a disease or condition.
  • Such diagnosis and detection can be accomplished by coupling an antibody to a detectable substance, including but not limited to a variety of enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactose Glycosidase or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent substances such as, but not limited to, umbelliferone, fluorescein, isothiocyanate , rhodamine, dichlorotriazinamide fluorescein, dansyl chloride or phycoerythrin; luminescent substances such as, but not limited to, luminol; bioluminescent substances such as, but not limited to, luciferase, luciferin and jellyfish Photoprotein; radioactive material such as, but not limited to, iodine ( 131 I, 125 I, 123 I and 121 I), carbon ( 14 C
  • the invention also encompasses the use of antibody molecules conjugated to a therapeutic moiety.
  • the antibody molecule can be conjugated to a therapeutic moiety, such as a cytotoxin (eg, a cytostatic or cytotoxic agent), a therapeutic agent, or a radioactive metal ion, such as an alpha emitter.
  • a cytotoxin eg, a cytostatic or cytotoxic agent
  • a therapeutic agent e.g, a cytostatic or cytotoxic agent
  • a radioactive metal ion such as an alpha emitter.
  • an antibody molecule of the invention can be conjugated to a therapeutic moiety or moiety that modulates a given biological response.
  • the therapeutic or drug moiety should not be construed as being limited to classical chemotherapeutics.
  • the drug moiety can be a protein, peptide or polypeptide possessing the desired biological activity.
  • Such proteins may, for example, include toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha interferon, beta interferon, nerve Growth factors, platelet-derived growth factors, tissue plasminogen activators, apoptotic agents, anti-angiogenic agents, or biological response modifiers, such as lymphokines.
  • toxins such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin
  • proteins such as tumor necrosis factor, alpha interferon, beta interferon, nerve Growth factors, platelet-derived growth factors, tissue plasminogen activators, apoptotic agents, anti-angiogenic agents, or biological response modifiers, such as lymphokines.
  • the antibody molecules of the invention can be conjugated to a therapeutic moiety such as a radioactive metal ion, such as an alpha-emitter such as 213 Bi or can be used to catalyze the emission of metal ions (including but not limited to 131 In, 131 LU, 131 Y, 131 Ho , 131 Sm) a macrocyclic chelating agent conjugated to the polypeptide.
  • a macrocyclic chelating agent is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), which can be passed through a linker The molecule attaches to the antibody.
  • linker molecules are well known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90, each of which is incorporated by reference in its entirety.
  • the antibody may also be linked to a solid support, which is particularly useful for immunoassays or purification of target antigens.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the antibody molecules of the invention can be obtained, for example, by solid peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production.
  • a polynucleotide encoding any one of the polypeptide chains and/or a plurality of polypeptide chains of the antibody molecule is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • the polynucleotide can be easily isolated and sequenced using conventional methods.
  • a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.
  • Expression vectors can be constructed using methods well known to those of skill in the art.
  • Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YAC).
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to accomplish this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, liposome-based transfection, or other conventional techniques.
  • a host cell comprising one or more polynucleotides of the invention.
  • a host cell comprising an expression vector of the invention.
  • the term "host cell” refers to any type of cellular system that can be engineered to produce an antibody molecule of the invention.
  • Host cells suitable for replicating and supporting expression of an antibody molecule of the invention are well known in the art. Such cells can be transfected or transduced with a specific expression vector, as desired, and a large number of cells containing the vector can be cultured for inoculating a large scale fermenter to obtain a sufficient amount of the antibody molecule of the present invention for clinical use.
  • Suitable host cells include prokaryotic microorganisms such as E.
  • coli eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • CHO Chinese hamster ovary cells
  • Mammalian cell lines suitable for suspension culture can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2/0.
  • the host cell is a CHO, HEK293 or NSO cell.
  • a method of producing an antibody molecule of the invention comprising culturing a host cell as provided herein under conditions suitable for expression of the antibody molecule, the host cell comprising the encoding A polynucleotide of an antibody molecule, and the antibody molecule is recovered from a host cell (or host cell culture medium).
  • Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art.
  • the purity of the antibody molecules of the present invention can be determined by any of a variety of well known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.
  • compositions for example, pharmaceutical compositions comprising an antibody molecule described herein formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutical compositions of the invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).
  • compositions of the invention may be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (for example, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions for example, injectable solutions and infusible solutions
  • dispersions or suspensions for example, liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • a common preferred composition is in the form of an injectable solution or an infusible solution.
  • a preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
  • the antibody molecule is administered by intravenous infusion or injection.
  • the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • parenteral administration and “parenteral administration” as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
  • compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes or lyophilized forms.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody molecule) in a suitable amount in a suitable solvent, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle containing base dispersion medium and other ingredients.
  • a coating agent such as lecithin or the like can be used.
  • the proper fluidity of the solution can be maintained by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by the inclusion in the compositions of the compositions which delay the absorption, such as the monostearate and gelatin.
  • an antibody molecule of the invention can be administered orally, for example, orally with an inert diluent or an edible carrier.
  • the antibody molecules of the invention may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets or incorporated directly into the subject's diet.
  • the compound can be incorporated with excipients and in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, glutinous rice papers It is used in the form of a wafer or the like.
  • Therapeutic compositions can also be administered using medical devices known in the art.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of an antibody molecule of the invention.
  • “Therapeutically effective amount” means an amount effective to achieve the desired therapeutic result at the desired dosage and for the period of time required.
  • the therapeutically effective amount can vary depending on various factors such as the disease state, the age, sex, and weight of the individual.
  • a therapeutically effective amount is any amount that is toxic or detrimental to a therapeutically beneficial effect.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (eg, a tumor growth rate) of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%.
  • the ability of an antibody molecule of the invention to inhibit measurable parameters e.g., tumor volume
  • prophylactically effective amount is meant an amount effective to achieve the desired prophylactic result at the desired dosage and for the period of time required. Generally, the prophylactic amount is less than the therapeutically effective amount since the prophylactic dose is administered to the subject prior to the earlier stage of the disease or at an earlier stage of the disease.
  • Kits comprising the antibody molecules described herein are also within the scope of the invention.
  • the kit may contain one or more additional elements including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
  • the antibody molecules disclosed herein have diagnostic and therapeutic and prophylactic uses in vitro and in vivo.
  • these molecules can be administered to cultured cells in vitro or ex vivo or to a subject, eg, a human subject, to treat, prevent, and/or diagnose a variety of antigen-related diseases, such as cancer, autoimmune diseases. , acute and chronic inflammatory diseases, infectious diseases (for example, chronic infectious diseases or sepsis).
  • the invention provides a diagnostic method for detecting a biological sample, such as serum, semen, or a urine or tissue biopsy sample (eg, from a hyperproliferative or cancerous lesion) in vitro or in vivo.
  • the diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with an antibody molecule as described herein or administering the antibody molecule to a subject, and (ii) allowing the interaction to occur.
  • the formation of a complex between the antibody molecule and the sample (and optionally, the control sample) is detected. Formation of the complex indicates the presence of a relevant antigen and may indicate the suitability or need for the treatment and/or prevention described herein.
  • the relevant antigen is detected prior to treatment, for example, prior to initiation of treatment or prior to treatment after the treatment interval.
  • Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (eg, RT-PCR), or in vivo imaging techniques.
  • antibody molecules used in in vivo and in vitro assays are labeled, directly or indirectly, with a detectable substance to facilitate detection of bound or unbound conjugates.
  • Suitable detectable materials include a variety of biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (eg, nuclear magnetic resonance) materials, and radioactive materials.
  • the level and/or distribution of the relevant antigen is determined in vivo, eg, in a non-invasive manner (eg, by detecting using a suitable imaging technique (eg, positron emission tomography (PET) scan)) Labeled antibody molecules of the invention.
  • a suitable imaging technique eg, positron emission tomography (PET) scan
  • PET positron emission tomography
  • the relevant antigen is determined in vivo, for example, by detecting an antibody molecule of the invention that is detectably labeled with a PET reagent (eg, 18 F-fluorodeoxyglucose (FDG)).
  • FDG F-fluorodeoxyglucose
  • the invention provides a diagnostic kit comprising the antibody molecule described herein and instructions for use.
  • the invention relates to the use of an antibody molecule of the invention in vivo for the treatment or prevention of a condition in which an immune response is modulated in a subject, thereby inhibiting or reducing related diseases such as cancerous tumors, autoimmune diseases, acute and chronic The onset or recurrence of inflammatory diseases, infectious diseases (eg, chronic infectious diseases or sepsis).
  • the antibody molecule of the present invention can be used alone.
  • the antibody molecule can be administered in combination with other cancer therapeutic/preventive agents.
  • the antibody molecule of the invention is administered in combination with one or more other drugs, such combinations can be administered in any order or simultaneously.
  • the invention provides a method of modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody molecule described herein.
  • the invention provides a method of preventing the onset or recurrence of a disease in a subject, the method comprising administering to the subject a prophylactically effective amount of an antibody molecule described herein.
  • cancers treated and/or prevented with antibody molecules include, but are not limited to, solid tumors, hematological cancers (eg, leukemias, lymphomas, myeloma, eg, multiple myeloma), and metastatic lesions.
  • the cancer is a solid tumor.
  • solid tumors include malignant tumors, for example, sarcomas and carcinomas of multiple organ systems, such as invasive lungs, breasts, ovaries, lymphoid, gastrointestinal (eg, colon), anal, genital, and genitourinary tract (eg, Kidney, bladder epithelium, bladder cells, prostate), pharynx, CNS (eg, brain, nerve or glial cells), head and neck, skin (eg, melanoma), nasopharynx (eg, differentiated or undifferentiated) Metastatic or locally recurrent nasopharyngeal carcinoma) and those of the pancreas, as well as adenocarcinomas, including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Cancer can be in early, intermediate or advanced stages or metastatic cancer.
  • the cancer is selected from the group consisting of melanoma, breast cancer, colon cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cancer (eg, renal cell carcinoma), liver cancer, non-small cell lung cancer (NSCLC) ), ovarian cancer, pancreatic cancer, prostate cancer, head and neck cancer, stomach cancer, hematological malignancies (eg, lymphoma).
  • GIST gastrointestinal stromal tumor
  • renal cancer eg, renal cell carcinoma
  • liver cancer eg, non-small cell lung cancer (NSCLC)
  • ovarian cancer pancreatic cancer
  • prostate cancer head and neck cancer
  • stomach cancer hematological malignancies
  • an infectious disease that is treated and/or prevented with an antibody molecule includes a pathogen that is currently in the absence of an effective vaccine or a pathogen to which a conventional vaccine is not fully effective.
  • pathogens include, but are not limited to, HIV, (A, B, and C) hepatitis, flu, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa.
  • the blocking effect of the exemplified antibody molecules of the present invention on PD-L1 is particularly useful for combating infections established by pathogens (e.g., HIV) in which a variant antigen occurs as the infection progresses.
  • variant antigens can be regarded as foreign antigens upon administration of an anti-human PD-L1 antibody, whereby the antibody molecules exemplified in the present invention are capable of eliciting a strong T cell response which is not inhibited by a negative signal by PD-L1.
  • the immune system is downregulated by treatment and/or prevention of inflammatory and autoimmune diseases and graft versus host disease (GvHD) with an antibody molecule of the invention.
  • autoimmune diseases that can be treated and/or prevented by administration of an antibody molecule of the invention include, but are not limited to, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, Crohn's disease, lupus erythematosus, ulcerative colitis, uveitis Wait.
  • inflammatory diseases that can be treated and/or prevented by administration of the antibody molecules of the invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, allergic diseases, septic shock, pulmonary fibrosis, arthritis, and chronic virality. Or chronic inflammation caused by bacterial infections.
  • Example 1 Construction, expression, purification and characterization of anti-OX40/PD-L1 bispecific antibody
  • bispecific antibody Bi-110-112HC and its structure is shown in FIG. 1A
  • Bispecific antibody Bi-113-112HC its structure is shown in Figure 1B
  • bispecific antibody Bi-119-112LC its structure is shown in Figure 1C
  • bispecific antibody Bi-122-112LC its structure is shown in Figure 1D.
  • the four anti-OX40/PD-L1 bispecific antibodies are described separately below.
  • the bispecific antibody Bi-110-112HC is composed of four polypeptide chains symmetrically bilaterally, wherein two polypeptide chains in the left half (ie, peptide chain #1 and peptide chain#) 2)
  • the VL amino acid sequence represented by SEQ ID NO: 7 derived from the anti-OX40 antibody ADI-20112 is contained in the peptide chain #1 shown in SEQ ID NO: 6 from the N-terminus to the C-terminus, in the VL a human kappa light chain constant region (CL) amino acid sequence represented by SEQ ID NO: 8 at the C-terminus of the amino acid sequence, and a linker peptide represented by SEQ ID NO: 9 at the C-terminus of the human kappa light chain constant region (CL) amino acid sequence
  • VH amino acid sequence represented by SEQ ID NO: 11 derived from the anti-OX40 monoclonal antibody ADI-20112, and the human IgG1 derived from the C-terminus of the VH amino acid sequence are contained in the peptide chain #2 shown in SEQ ID NO:
  • the bispecific antibody Bi-113-112HC is composed of four polypeptide chains symmetrically bilaterally, wherein two polypeptide chains in the left half (ie, peptide chain #1 and peptide chain#) 2)
  • the anti-PD-L1 VHH amino acid sequence represented by SEQ ID NO: 2 and the linker peptide represented by SEQ ID NO: 9 are included in the peptide chain #1 shown in SEQ ID NO: 14 from the N-terminus to the C-terminus.
  • amino acid sequence The amino acid sequence, the VL amino acid sequence derived from the anti-OX40 antibody ADI-20112 shown in SEQ ID NO: 7, and the human kappa light chain constant region (CL) amino acid sequence set forth in SEQ ID NO: 8.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 10.
  • the bispecific antibody Bi-119-112LC is composed of four polypeptide chains symmetrically bilaterally, wherein two polypeptide chains in the left half (ie, peptide chain #1 and peptide chain#) 2)
  • the amino acid sequence shown by SEQ ID NO: 15 and SEQ ID NO: 16 from the N-terminus to the C-terminus, respectively.
  • the VL amino acid sequence derived from the anti-OX40 antibody ADI-20112 represented by SEQ ID NO: 7 and the SEQ ID NO: are included from the N-terminus to the C-terminus in the peptide chain #1 shown in SEQ ID NO: 15.
  • the N-terminal to C-terminus of the peptide chain #2 shown in SEQ ID NO: 16 comprises the VH amino acid sequence derived from the anti-OX40 monoclonal antibody ADI-20112 represented by SEQ ID NO: 11, and the CH1 derived from human IgG1.
  • the bispecific antibody Bi-122-112LC is composed of four polypeptide chains symmetrically left and right, and two polypeptide chains in the left half (ie, peptide chain #1 and peptide chain#) 2) having an amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 17 from the N-terminus to the C-terminus, respectively, wherein the peptide chain #2 comprises the SEQ ID NO: 2 from the N-terminus to the C-terminus.
  • the anti-PD-L1 VHH amino acid sequence, the linker peptide amino acid sequence shown in SEQ ID NO: 9, the VH amino acid sequence derived from the anti-OX40 monoclonal antibody ADI-20112 shown in SEQ ID NO: 11, and the SEQ ID NO: 12 The CH1 amino acid sequence derived from human IgG1 and the amino acid sequence derived from the human IgG1 Fc region set forth in SEQ ID NO: 13.
  • Example 1.2 Expression, purification and analysis of anti-OX40/PD-L1 bispecific antibodies
  • nucleotide sequences encoding the peptide chain #1 and peptide chain #2 of the anti-OX40/PD-L1 bispecific antibody constructed in Example 1.1 were respectively ligated into the commercially available true
  • the nuclear expression vector pTT5 was expressed and purified in eukaryotic cells, and anti-OX40/PD-L1 bispecific antibodies Bi-110-112HC, Bi-113-112HC, Bi-119-112LC and Bi-122-112LC were obtained. .
  • the specific operation is as follows.
  • the gene encoding the above-mentioned peptides of the bispecific antibodies Bi-110-112HC, Bi-113-112HC, Bi-119-112LC and Bi-122-112LC was synthesized by Genewiz. sequence.
  • the nucleotide sequence encoding the synthesized peptide chain was ligated into the vector pTT5 using a suitable restriction enzyme and ligase, respectively, to obtain a recombinant vector containing the nucleotide sequence encoding the peptide chain, respectively.
  • the recombinant vector was verified by sequencing and used for subsequent expression.
  • HEK293 cells (purchased from Invitrogen) were subcultured in Expi293 cell culture medium (purchased from Invitrogen). The cell culture was centrifuged one day before the transfection to obtain a cell pellet, and the cells were suspended with fresh Expi293 cell culture medium to adjust the cell density to 1 ⁇ 10 6 cells/ml. The HEK293 cells were further cultured so that the cell density in the culture on the day of transfection was about 2 x 10 6 cells/ml. A final volume of HEK293 cell suspension of 1/10 F17 medium (purchased from Gibco, Cat. No. A13835-01) was used as a transfection buffer.
  • the culture flask was supplemented with FEED (Sigma, catalog number: H6784-100G) at a concentration of 1/50 of the culture volume after transfection and a concentration of 1/50 of the culture volume after transfection. 200 g / L of glucose solution, gently mixed, placed in 8% CO 2 , 36.5 ° C continue to culture. After 20 hours, VPA (Gibco, catalog number: 11140-050) was added to a final concentration of 2 mM/L.
  • FEED Sigma, catalog number: H6784-100G
  • the specific affinity chromatography purification step is: using MabSelect SuRe (GE Healthcare, catalog number: 17-5438-03) affinity chromatography column, and placed in the AKTApure system.
  • the AKTApure system equipped with a MabSelect SuRe affinity chromatography column was detoxified overnight with 0.1 M NaOH, and then the system was washed with 5 column volumes of binding buffer (Tris 20 mM, NaCl 150 mM, pH 7.2) and the column was equilibrated. The supernatant of the above filtered cells was passed through a column. The cells were re-equilibrated with 5 to 10 column volumes of binding buffer and monitored for UV-leveling using an ultraviolet detection device equipped with an AKTApure system.
  • the antibody was eluted with an elution buffer (citric acid + sodium citrate 100 mM, pH 3.5), and samples were collected according to the ultraviolet absorption value. Each 1 ml of the collection solution was neutralized by adding 80 ⁇ l of a neutralization buffer (Tris-HCl 2M).
  • the purity of the samples in the collected fractions was measured by size exclusion chromatography (SEC).
  • SEC results are shown in Fig. 2A, Fig. 2B, Fig. 2C and Fig. 2D, respectively.
  • the purity of the bispecific antibody Bi-110-112HC is 71.40%
  • the purity of Bi-113-112HC is 84.54%
  • the purity of Bi-119-112LC is 99.43. %
  • Bi-122-112LC purity was 94.79%.
  • the purified bispecific antibody solution was centrifuged at 4500 rpm for 30 minutes using a 15 ml ultrafiltration centrifuge tube.
  • the protein was diluted with PBS, centrifugation was continued, and centrifugation was performed at 4500 rpm for 30 minutes, and the operation was repeated twice to replace the buffer.
  • the antibodies after buffer exchange were combined and the antibody concentration was measured.
  • the equilibrium dissociation constant (K D ) of the above-described exemplary anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention in combination with OX40 and PD-L1 was determined by a kinetic binding assay using an Octet system (manufactured by ForteBio) . ).
  • the ForteBio affinity assay was performed according to the method reported in the literature (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5(2): p. 270-278). Briefly, AHC sensor (Pall, Cat. No.
  • the anti-human IgG Fc biosensor AHC was immersed in wells containing the antibody solution, respectively, and immersed at room temperature for 600 seconds. The sensor was then washed in SD buffer until baseline was reached and then immersed in wells containing 100 ⁇ l of antigen solution to monitor binding of the antibody to the antigen. The sensor was then transferred to a well containing 100 ⁇ l of SD buffer to monitor antibody dissociation. The speed was 400 rpm and the temperature was 30 °C. The background corrected binding curves and dissociation curves were fitted by Octet analysis software (ForteBio) to generate binding (k on ) and dissociation (k dis ) rate constants which were subsequently used to calculate the equilibrium dissociation constant (K D ). The on , k dis and K D data of the bispecific antibody Bi-119-112LC and the antigen OX40 or PD-L1 are shown in Tables 1 and 2.
  • the bispecific antibody Bi-119-112LC of the present invention is capable of simultaneously binding to PD-L1 and OX40 proteins in solution, and maintains the parent antibody ADI-20112 and humanized Nb-Fc and each corresponding antigen. Affinity constant.
  • Example 1.4 Binding analysis of anti-OX40/PD-L1 bispecific antibody of the present invention to CHO cells overexpressing OX40 or PD-L1
  • Binding of the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention to CHO cells overexpressing OX40 or PD-L1 was measured by FACS.
  • ExpiCHO TM Expression System Kit (Invitrogen, catalog number: A29133), according to the manufacturer's instructions embodiment operates as follows: carrying cloned into the multiple cloning site MCS human PD-L1 cDNA (Sino Biological Inc. ) of The pCHO1.0 vector (Invitrogen) was transfected into Chinese hamster ovarian cancer cells (CHO) (Invitrogen) to produce CHO cells (CHO-PD-L1 cells) overexpressing human PD-L1. CHO-PD-L1 cells were counted, diluted to 1 ⁇ 10 6 cells/ml with a cell culture medium, and added to a U-bottom 96-well plate at 100 ⁇ l/well.
  • the cell culture medium was removed by centrifugation at 400 g for 5 minutes on a centrifuge.
  • 100 ⁇ l of the serially diluted bispecific antibody Bi-119-112LC of the present invention and the humanized Nb-Fc as a control were separately added to the U-shaped plate and the cells were resuspended and allowed to stand on ice for 30 minutes.
  • the supernatant was removed, and unbound antibody was removed by washing the cells with PBS.
  • 100 ⁇ l of 1:200 diluted PE-conjugated anti-human Fc antibody (SOUTHERN BIOTECH) was added to each well and incubated on ice for 30 minutes in the dark.
  • the bispecific antibody Bi-119-112LC of the present invention is capable of binding to PD-L1 expressed on the cell surface, and has an EC50 of 2.654 nM, and PD-L1 expressed on the cell surface with the parent anti-PD-L1 antibody.
  • the binding capacity (EC50 is 1.940nM) is similar.
  • overexpression of human OX40 was generated by transfecting the human OX40 cDNA (Invitrogen) carrying human OX40 cDNA (Sino Biological Inc.) cloned into the multiple cloning site MCS into Chinese hamster ovarian cancer cells (CHO) (Invitrogen). CHO cells (CHO-OX40 cells).
  • FACS detection was performed on CHO-OX40, except that the cells used were different and the ADI-20112 antibody was used as the control antibody, and the other experimental procedures were the same as those of the CHO-PD-L1 cells described above.
  • the bispecific antibody Bi-119-112LC of the present invention is capable of binding to OX40 expressed on the cell surface, and has an EC50 of 3.195 nM, and the binding ability of the parent anti-OX40 antibody to OX40 expressed on the cell surface (EC50 is 2.193nM) is similar.
  • Example 1.5 Analysis of the anti-OX40/PD-L1 bispecific antibody of the present invention simultaneously binding to CHO cells overexpressing OX40 and CHO cells overexpressing PD-L1 -
  • the present embodiment detects the bispecific antibody by flow cytometry. Induction of different cell cross-linking conditions.
  • the specific experimental process is as follows.
  • CHO-PD-L1 cells and CHO-OX40 cells were obtained and cultured as described in Example 1.4. Cultures containing CHO-PD-L1 cells and CHO-OX40 cells were separately centrifuged at 400 g for 5 minutes on a centrifuge to remove the cell culture medium. After washing once with PBS, the cells were resuspended in PBS. The cells were counted and the cell density was adjusted to 2 x 10 6 cells/ml. The CHO-PD-L1 cells, and CHO-OX40 cells were 1: 5000 was added CellTracker TM Deep Red (Thermo) and Cell Trace CFSE (Invitrogen) dye, placed in 37 °C 30 minutes. The cells were centrifuged at 400 g for 5 minutes on a centrifuge, the supernatant was removed, and the cells were washed once with PBS.
  • TM Deep Red Thermo
  • Cell Trace CFSE Invitrogen
  • the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC was able to induce cross-linking of CHO-PD-L1 cells and CHO-OX40 cells, thereby indicating the bispecificity of the present invention.
  • Antibodies are capable of binding to target antigens from different cell surfaces simultaneously.
  • the heavy chain (HC) amino acid sequence of the IgG1 negative control used in this example is shown in SEQ ID NO: 29; the light chain (LC) amino acid sequence of the IgG1 negative control is shown in SEQ ID NO:30.
  • Anti-OX40/PD-L1 bispecific antibody of the invention blocks binding of PD-1 to CHO cells overexpressing PD-L1
  • this example detects the anti-OX40/PD of the present invention by flow cytometry.
  • the -L1 bispecific antibody blocks the binding of PD-1 protein to CHO cells overexpressing PD-L1.
  • CHO-PD-L1 cells were obtained and cultured as described in Example 1.4.
  • the culture containing 2.4 ⁇ 10 7 CHO-PD-L1 cells was centrifuged at 400 g for 5 minutes on a centrifuge to remove the cell culture medium. After washing once with PBS, the cells were resuspended in 5 ml of PBS.
  • the IgG1 negative control used in this example was the same as the IgG1 negative control used in Example 1.5 above.
  • Experimental Results As shown in Fig. 6 , the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention can effectively block the binding of PD-1 to CHO cells overexpressing PD-L1, and block the activity and
  • the anti-PD-L1 humanized Nb-Fc antibody was similar (the IC50 of the anti-OX40/PD-L1 bispecific antibody was 3.522 nM, and the IC50 of the anti-PD-L1 humanized Nb-Fc antibody was 4.906 nM).
  • Example 1.7 Detection of anti-PD-L1 activity of anti-OX40/PD-L1 bispecific antibody based on luciferase reporter gene
  • this example uses a luciferase reporter gene detection cell line (Promega, CS187109) By detecting the expression of luciferase, the bispecific antibody inhibits the interaction of PD-1/PD-L1 interaction.
  • a luciferase reporter gene detection cell line Promega, CS187109
  • this example uses PD-1/PD-L1 Blockade Bioassay, Cell Propagation Model (Promega) The anti-PD-L1 biological activity of the bispecific antibody of the present invention was investigated.
  • Promega's PD-1/PD-L1 Blockade Bioassay is a biologically relevant MOA-based assay for determining the potency and stability of antibodies that block PD-1/PD-L1 interaction.
  • the assay consists of two genetically engineered cell lines:
  • PD-1 effector cells Stable expression of human PD-1 and Jurkat T cells expressing luciferase by a nuclear factor of activated T cells (NFAT).
  • PD-L1 aAPC/CHO-K1 cells CHO-K1 cells stably expressing human PD-L1 and cell surface proteins that activate the corresponding TCR in an antigen-independent manner.
  • PD-1 binds to PD-L1 to block the transduction of NFAT downstream signals, thereby inhibiting the expression of luciferase.
  • PD-1 antibody or PD-L1 antibody is added, this blocking effect is reversed.
  • the photozyme is expressed to thereby detect a fluorescent signal.
  • the detection method has good sensitivity, specificity and accuracy, and the stability is very good.
  • PD-L1 aAPC/CHO-K1 cells were plated one day before the activity test: the culture supernatant was discarded, washed once with PBS, trypsin (Gibco, 25200072), incubated at 37 ° C for 3-5 min, using four volumes of 10
  • the RPMI1640 (Gibco, 22400-071) medium of %FBS (HyClone, SH30084.03) was digested, the cells were collected, and a small amount of cell mixture was taken to determine the cell concentration. The required volume of cell fluid was taken, 400 g, centrifuged for 10 min, discarded.
  • the cells were resuspended in RPMI 1640 (Gibco, 22400-071) medium containing 10% FBS (HyClone, SH30084.03) as an assay buffer so that the cell density was 4 ⁇ 10 5 cells/ml.
  • the cell suspension was added to a 96-well white cell culture plate (Nunclon, 136101) at 100 ⁇ L/well, and a side hole of a 96-well white cell culture plate was added to PBS at 200 ⁇ l/well.
  • the cells were cultured overnight in a carbon dioxide incubator at 37 ° C in a 5% CO 2 incubator.
  • PD-1 effector cells were taken, counted, 400 g, centrifuged for 5 min, and the cells were resuspended in an assay buffer so that the cell concentration was 1.25 ⁇ 10 6 cells/ml.
  • the IgG1 negative control used in this example was the same as the IgG1 negative control used in Example 1.5 above.
  • Experimental Results As shown in Figure 7, the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention can effectively abolish the blocking effect of PD1/PD-L1 interaction on the NFAT signaling pathway, and the activity and anti-PD
  • the -L1 humanized Nb-Fc antibody was similar (the EC50 of the anti-OX40/PD-L1 bispecific antibody was 0.4585 nM, and the EC50 of the anti-PD-L1 humanized Nb-Fc antibody was 0.3283 nM).
  • the OX40-mediated signaling pathway biological activity was activated in the presence of CHO-PD-L1 cells as described in Example 1.4.
  • This example uses the Jurkat-OX40-NFkB-Luc-Rep stable cell line of Cinda Biopharmaceutical (Suzhou) Co., Ltd. to measure OX40-mediated transcriptional activation to evaluate whether the anti-OX40/PD-L1 bispecific antibody of the present invention is Has activator activity against the OX40 antibody.
  • Human OX40 construct purchased from Sino
  • NFkB Human OX40 construct (purchased from Sino) and NFkB were introduced by anti-human CD3 (BD Biosciences, catalog number: 555329), anti-human CD28 (BD Biosciences, catalog number: 555725) plus the antibody of the present invention in solution.
  • - luciferase construct promoter of NFkB -luc, Promega
  • human OX40 overexpressing Jurkat cells obtained from the American ATCC for 16 hours, followed by addition of Bio-Glo TM reagent color.
  • the specific experimental process is as follows:
  • Solution preparation Analytical buffer: RPIM-1640 (90%) (Gibco, 22400-071), FBS (10%) (HyClone, SH30084.03), anti-human CD3 (2 ⁇ g/ml) (BD Biosciences, catalog number: 555329), anti-human CD28 (2 ⁇ g/ml) (BD Biosciences, catalog number: 555725), now available.
  • the IgG1 negative control used in this example was the same as the IgG1 negative control used in Example 1.5 above.
  • the results of the experiment are shown in Figure 8.
  • the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention has a significant activation of the NFkB signaling pathway, while the anti-OX40 Antibody ADI-20112 detected a lower NFkB signaling pathway activation effect, and anti-PD-L1 humanized Nb-Fc antibody did not have an NFkB signaling pathway activation effect.
  • the anti-OX40/PD-L1 bispecific antibody of the present invention shows that the NFkB signaling pathway downstream of OX40 can be better activated in the presence of PD-L1 expressing cells.
  • Differential scanning fluorimetry provides information about the structural stability of a protein based on the fluorescence changes in the protein profile, detects changes in the conformation of the protein, and obtains the melting temperature (T m ) of the protein.
  • T m melting temperature
  • the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC antibody of the present invention was diluted to 1 mg/ml with a PBS solution, respectively.
  • SYPRO Orange Protein Gel Stain (Gibco, catalog number: S6650)
  • 196 ⁇ l of PBS was added, and SYPRO Orange Protein Gel Stain was diluted 50-fold.
  • the bispecific antibody of the present invention has a T m of >60 ° C and, therefore, has good thermal stability.
  • the present example evaluated the antibody by measuring the change in purity of the prepared antibody at 40 ° C for 0, 1, 3, 7, 10, 20, and 30 days. Long-term thermal stability.
  • the initial purity of the prepared batch of Bi-119-112LC antibody was 92.91% as determined by SEC.
  • the experimental method was as follows: The antibody sample was concentrated to 5 mg/ml (dissolved in PBS), dispensed in an EP tube, 200 ⁇ l/tube, and placed at 40 ° C in the dark. One tube was taken on days 0, 1, 3, 7, 10, 20, and 30, and the purity of the main peak of the monomer was measured by SEC-HPLC.
  • the experimental results are shown in Table 4.
  • the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention was allowed to stand at 40 ° C for 30 days, and the ratio of the main peak of the monomer was reduced by only 3.69%.
  • the results indicate that the anti-OX40/PD-L1 bispecific antibody of the present invention has good thermal stability.
  • Example 1.11 Detection of activation of human CD4 + T cells by anti-OX40/PD-L1 bispecific antibody of the present invention
  • This example detects the activation of human CD4 + T cells by anti-OX40/PD-L1 bispecific antibody in vitro.
  • the detailed experimental procedure is as follows:
  • PBMC cells Resuscitate human PBMC cells (ALLCELLS, PB005F), the cells after standing for 3 hours are mononuclear cells, add 10ml AIM Medium CTS (GIBCO, A3021002) medium, adding IL4 (20ng/ml) (R&D, 204-IL), GM-CSF (10ng/ml) (R&D, 215-GM) to induce monocyte differentiation into dendritic cells (ie, DC cells), cultured until day 5, adding cytokine TNF ⁇ (1000 U/ml) that induces DC maturation (R&D, catalog number: 210-TA), RhIL-1 ⁇ (5 ng/ml) (R&D, catalog number :201-LB), RhIL-6 (10 ng/ml) (R&D, catalog number: 206-IL), 1 ⁇ M PGE (Tocris, catalog number: 2296), in a carbon dioxide incubator at 37 ° C, 5% CO 2 culture conditions Continue to culture for 2 days as mature DC cells (mo
  • CD4 + T cells were cultured in Medium CTS medium.
  • Add CD4 + T cells: anti-CD3/CD28 beads 1:1 to Dynabeads Human T-Activator CD3/CD28 (INVITROGEN, catalog number: 11131D), culture in a carbon dioxide incubator at 37 ° C, 5% CO 2 culture conditions 3 Days, performing bead stimulation on CD4 + T cells;
  • the above isolated DC cells were mixed with bead-stimulated CD4 + T cells, and added to the Staphylococcal enterotoxin E superantigen (Toxin technology, catalog number: ET404) at a final concentration of 1 ng/ml, 200 ⁇ l per well, and 12,000 DC cells.
  • 120,000 CD4 + T cells were added to the diluted antibody for 3 days.
  • the expression of IL2 in each sample was detected by Cisbio IL2 detection kit (CISBIO, catalog number: 62HIL02PEG), and the expression of IL2 in different antibodies was reflected. The ability of the antibody to activate T cells.
  • the anti-OX40/PD-L1 bispecific antibody Bi-119-112LC of the present invention can effectively activate human CD4 + T cells in vitro, and its activation effect is stronger than that of anti-PD-L1 humanized Nb-Fc.
  • the antibody, anti-OX40 antibody ADI-20112 is stronger.
  • bispecific antibody Bi-2-50 the structure of which is shown in Figure 11A
  • the bispecific antibody Bi-2-51 has a structural schematic diagram as shown in Fig. 11B.
  • the two anti-VEGF/GITR bispecific antibodies are described separately below.
  • the bispecific antibody Bi-2-50 is composed of four polypeptide chains symmetrically left and right, and two polypeptide chains in the left half (i.e., peptide chain #1 and peptide chain#) 2)
  • the VL amino acid sequence represented by SEQ ID NO: 20 derived from the anti-VEGF antibody Avastin and the SEQ ID NO at the C-terminus of the VL amino acid sequence are contained in the peptide chain #1 shown in SEQ ID NO: 18.
  • the human kappa light chain constant region (CL) amino acid sequence shown in Figure 8 comprising the VH amino acid set forth in SEQ ID NO: 22 derived from the anti-VEGF monoclonal antibody Avastin in the peptide chain #2 set forth in SEQ ID NO:21. a sequence, a CH1 amino acid sequence represented by SEQ ID NO: 23 derived from human IgG1 at the C-terminus of the VH amino acid sequence, a linker peptide amino acid sequence represented by SEQ ID NO: 9 at the C-terminus of the CH1 amino acid sequence, and SEQ ID The anti-GITR VHH amino acid sequence shown by NO: 24, and the Fc region amino acid sequence derived from human IgG1 shown by SEQ ID NO: 13.
  • the bispecific antibody Bi-2-51 is composed of four polypeptide chains symmetrically left and right, and two polypeptide chains in the left half (i.e., peptide chain #1 and peptide chain#) 2) The amino acid sequences shown in SEQ ID NO: 18 and SEQ ID NO: 28, respectively, from the N-terminus to the C-terminus.
  • the N-terminal to C-terminus of the peptide chain #2 shown in SEQ ID NO: 28 comprises the VH amino acid sequence derived from the anti-VEGF monoclonal antibody Avastin represented by SEQ ID NO: 22, and the SEQ ID NO: 23
  • Example 2.2 Expression, purification and analysis of anti-VEGF/GITR bispecific antibodies
  • nucleotide sequences encoding the anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51 constructed in Example 2.1 were respectively passed through the peptide sequences #1 and # ⁇ #2.
  • the multiple cloning site was ligated into the commercially available eukaryotic expression vector pTT5, and expressed and purified in eukaryotic cells to obtain anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51.
  • the anti-VEGF/GITR bispecific antibody After purification, the anti-VEGF/GITR bispecific antibody has good purity, and the main peak purity of the bispecific antibodies Bi-2-50 and Bi-2-51 are 99.57% and 99.48%, respectively.
  • an antibody having the amino acid sequence of SEQ ID NO: 31 named "hcIgG-10", which comprises the SEQ ID NO: 24 from the N-terminus to the C-terminus is used.
  • the anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51 of the present invention were able to simultaneously bind VEGF165 (R&D, 293-VE-500) in solution. Binding to the GITR (AcroBiosystems, GIR-H5228-1MG) protein and maintaining the affinity constant of the parental antibody Avastin or hcIgG-10.
  • Example 2.4 Binding analysis of anti-VEGF/GITR bispecific antibodies of the invention with CHO cells overexpressing VEGF or GITR
  • Binding of the anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51 of the present invention to CHO cells overexpressing VEGF or GITR was measured by FACS.
  • the other specific experimental procedures were the same as in the above Example 1.4 except that the antibody and antigen used were different.
  • the IgG1 negative control used in this example was the same as the IgG1 negative control used in Example 1.5 above. The result is shown in FIG.
  • the anti-VEGF/GITR bispecific antibodies Bi-2-50 and Bi-2-51 of the present invention were both able to bind to GITR expressed on the cell surface with binding EC50 of 2.990 nM and 3.168 nM, respectively.
  • the EC50 of the parental antibody hcIgG-10 binding to the cell surface GITR was 0.6061 nM.

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Abstract

本发明提供了一种新型的经人工设计的抗体分子，其包含 (i)单结构域抗原结合位点； (ii)结合抗原的Fab片段；其中所述(i)位于所述(ii)的轻链可变结构域(VL)的N端或轻链恒定区(CL)的C端，或者所述(i)位于所述(ii)的重链可变结构域(VH)的N端或免疫球蛋白CH1结构域的C端，且所述(i)和(ii)分别结合相同或者不同的抗原，所述(i)和(ii)之间具有或者不具有连接肽。和位于所述(i)和(ii)的C端的(iii)免疫球蛋白Fc结构域。本发明还提供了编码所述抗体分子的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、包含所述抗体分子的免疫缀合物和药物组合物、以及所述抗体分子在疾病的免疫治疗、预防和/或诊断上的用途。

Description

新型抗体分子、其制备方法及其用途 发明领域
本发明总体上涉及免疫学和抗体工程领域。具体而言，本发明涉及多种新型的经人工设计的抗体分子、编码所述抗体分子的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、包含所述抗体分子的免疫缀合物和药物组合物、以及所述抗体分子在疾病的免疫治疗、预防和/或诊断上的用途。
发明背景
抗体分子能够与其相应的抗原发生靶向性的特异性结合，正日益成为针对各种疾病(例如，癌症、自身免疫病、炎性疾病、感染性疾病等)的重要的治疗剂、预防剂和/或诊断剂。但是，仅针对一种靶点的单特异性抗体在临床应用上存在一些局限性。患者在接受单特异性抗体治疗后可能产生耐药性或无应答。随着对癌症和其他多种疾病的研究，认识到了往往有多种信号转导通路参与疾病的发生和发展，单一靶点的免疫疗法在许多疾病中通常并不足以发挥对疾病的治疗作用。
由于多特异性抗体(例如，双特异性抗体)能够特异性结合不同抗原，当一种抗原位于特定的免疫细胞上，另一种抗原位于疾病细胞上时，多特异性抗体(例如，双特异性抗体)可以将特定的免疫细胞重新定向至疾病细胞，以增强免疫细胞对疾病细胞的杀伤力。另外，多特异性抗体(例如，双特异性抗体)也能够设计为同时作用于两种或多种不同介质的信号转导通路。这些优势特性为多特异性抗体(例如，双特异性抗体)开辟了广阔的应用前景。
已经通过抗体工程开发了大量富于想象力的多特异性抗体(例如，双特异性抗体)样式并且研究了它们在疾病应用上的适用性(Brinkmann U.和Kontermann R.E.,The making of bispecific antibodies，Mabs,2017,9(2):182-212)。目前，批准上市的2个双特异性抗体产品分别是Micromet公司和Amgen公司开发的Blinatumomab以及Trion Pharma公司开发的Catumaxomab。Blinatumomab是第1个在美国获批上市的用于治疗B细胞非霍奇金淋巴瘤(NHL)和B前体急性淋巴细胞白血病(ALL)的一种分子量约55KDa的单链双特异性抗体，由分别针对CD19分子和针对CD3分子的两种单链Fv分子通过柔性连接肽融合而成，其利用几乎在所有的B淋巴细胞肿瘤中都表达的CD19和在T细胞上表达的CD3，使T细胞与靶向细胞(肿瘤细胞)紧密联结在一起，T细胞释放穿孔素和粒端酶进入突触间隙，引起肿瘤细胞发生一系列化学反应，从而消灭肿瘤细胞(Nagorsen D.和Baeuerle P.A.,Immunomodulatory therapy of cancer with T cell-engaging BiTE antibody blinatumomab，Exp Cell Res，2011，317：1255-1260)。Catumaxomab是由两个分别源自亲本小鼠IgG2a同种型和大鼠IgG2b同种型的半抗体组成的嵌合体，其中每个半抗体具有一条轻链和一条重链，抗CD3大鼠IgG2b半抗体用于T细胞识别，抗肿瘤细胞表面抗原EpCAM(上皮细胞黏附分子)的小鼠IgG2a半抗体用于肿瘤细胞识别(Chelius D 等人，Structural and functional characterization of the trifunctional antibody catumaxomab，MAbs，2010，2:309-319)。Catumaxomab
Figure PCTCN2019079671-appb-000001
于2009年4月在欧洲获准用于治疗由EpCAM阳性上皮源性转移瘤所引起的恶性腹水。
多特异性抗体(例如，双特异性抗体)根据不同的组成部分以及构建方式，可以分为许多种类。例如，根据多特异性抗体结构的左右基本上对称性，可分为对称结构和不对称结构；根据多特异性抗体有无IgG的Fc区，可分为有Fc区的抗体样式和无Fc区的抗体样式；根据多特异性抗体中抗原结合位点的数量可分为二价、三价、四价或更多价的抗体等。
现有技术中的多特异性抗体样式在制备和应用中各有利弊，例如，虽然Blinatumomab可以通过重组中国仓鼠卵巢(CHO)细胞进行大规模培养生产，但是容易形成聚集物、在体内半衰期很短，实际使用的时候需要额外配备连续输液装置；Catumaxomab生产工艺复杂且鼠异源抗体比较容易在人体产生免疫原性问题。
因此，本领域仍然需要可供选择的具有改善性能的多特异性抗体样式。本发明提供了一种新的多特异性抗体样式，所述抗体样式通过使用分子量较小且稳定性高的单结构域抗原结合位点作为一种构建模块，与Fab片段的N端或C端连接，将所得的连接物再连接Fc区后，易于在体外的培养细胞中有效表达，不需要复杂的生产工艺；另外，在本发明抗体样式中Fc区的存在使得在培养细胞中表达本发明的抗体后，使用单步亲和层析即可获得经纯化的抗体，并且本发明的抗体在体内具有较长的血清半寿期并能引发效应子功能，诸如抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)和补体依赖的细胞毒作用(CDC)。本发明的多特异性抗体样式能够保持该多特异性抗体中的各抗原结合位点与相应的不同表位结合的亲和力，且在结合不同表位的时候互相之间不会产生空间位阻的干扰，具有好的成药性。进一步地，本发明的多特异性抗体样式是物理稳定的和生物学稳定的，这允许该抗体具有更好的生产性和可发展性。
发明概述
本文公开了通过抗体工程方法构建的一种新型的抗体分子。所述抗体分子能够以高亲和力和高特异性与一种或多种抗原结合，优选地，与两种以上的抗原结合。本发明还提供了编码所述抗体分子的核酸分子、用于产生所述抗体分子的表达载体、宿主细胞和方法。本发明还提供了包含本发明抗体分子的免疫缀合物和药物组合物。本文公开的抗体分子可以单独或与其他药物或其他治疗模式联合用来治疗、预防和/或诊断疾病，如自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)、肿瘤等。
因此，在一个方面，本发明提供了具有以下一个或多个特性的抗体分子：
(a)以高亲和力，例如以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与一种或多种抗原特异性结合；
(b)易于在体外的培养细胞中表达，且抗体分子的各条链之间能够正确偶合或配对；
(c)具有良好的物理稳定性，特别地，具有良好的长期热稳定性；且能长时间保持生 物学活性；和
(d)在与一种或多种抗原特异性结合后，通过调节(例如，抑制或者激活)各抗原所参与的信号传导通路来发挥生物学功能；
(e)在与一种或多种抗原特异性结合后，通过Fc区发挥效应子功能。
在一个实施方案中，本发明的抗体分子包含(i)单结构域抗原结合位点；(ii)结合抗原的Fab片段；其中所述(i)位于所述(ii)的轻链可变结构域(VL)的N端或轻链恒定区(CL)的C端，或者所述(i)位于所述(ii)的重链可变结构域(VH)的N端或免疫球蛋白CH1结构域的C端，且所述(i)和(ii)分别结合相同或者不同的抗原，所述(i)和(ii)之间具有或者不具有连接肽；以及位于所述(i)和(ii)的C端的(iii)免疫球蛋白Fc结构域。
在一个实施方案中，本发明的抗体分子包含至少四个抗原结合位点，分别是至少两个单结构域抗原结合位点和至少两个Fab片段中的抗原结合位点，结合至少四种、三种、两种不同抗原，或者结合一种相同抗原。对于所结合的每一种抗原而言，本发明抗体分子中的抗原结合位点结合的是抗原分子中的相同或不同表位。在一个实施方案中，本发明的抗体分子包含四个抗原结合位点，其中两个单结构域抗原结合位点结合第一抗原中的相同或不同表位，两个Fab片段结合第二抗原中的相同或不同表位，所述第一抗原不同于所述第二抗原。
在一个实施方案中，本发明抗体分子中的(i)和(ii)之间具有单独或组合使用的甘氨酸和/或丝氨酸残基作为连接肽，例如，所述连接肽包含氨基酸序列(Gly 4Ser)n，其中n是等于或大于1的正整数，例如，n是1－7中的正整数，例如，n是2，3，4，5，6。
在一个实施方案中，本发明抗体分子中的单结构域抗原结合位点选自重链可变结构域(VH)、轻链可变结构域(VL)、天然缺乏轻链的抗体的重链可变结构域(例如，骆驼科(Camelidae)物种中天然存在的重链抗体的重链可变结构域)、鱼类中称为新型抗原受体(new antigen receptor,NAR)的免疫球蛋白(如鲨鱼血清中天然存在的IgNAR)中的VH样单结构域、和衍生自它们的经重组的单结构域抗原结合位点(例如，骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域)。在一个优选的实施方案中，本发明抗体分子中的所述单结构域抗原结合位点选自骆驼科物种中天然存在的重链抗体的重链可变结构域、骆驼化的人VH结构域和人源化的骆驼科抗体重链可变结构域。将从天然缺乏轻链的重链抗体衍生的重链可变结构域在本文中也简称为VHH，以将其与四链免疫球蛋白的常规VH区分开。这种VHH分子可以衍生自骆驼科物种(例如骆驼、羊驼、单峰驼、驼羊和原驼)中产生的抗体。除骆驼科之外的其他物种也可以产生天然缺乏轻链的重链抗体，这类VHH也处于本发明的范围内。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链以及位于所述免疫球蛋白轻链可变结构域(VL)的N端的单结构域抗原结合位点，例如VHH；第二多肽链和第四多肽链中的每一多肽链包含免疫球蛋白重链。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链以及位于所述免疫球蛋白轻链恒定区(CL)的C端的单结构域抗原结合位点，例如VHH；第二多肽链和第四多肽链中的每一多肽链包含免疫球蛋白重链。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链；第二多肽链和第四多肽链中的每一多肽链包含免疫球蛋白重链以及位于所述免疫球蛋白重链N端的单结构域抗原结合位点，例如VHH。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变区、免疫球蛋白CH1结构域、单结构域抗原结合位点(例如VHH)、免疫球蛋白CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白重链可变结构域(VH)和免疫球蛋白轻链恒定区(CL)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1、CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)和单结构域抗原结合位点(例如VHH)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1、CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)和免疫球蛋白轻链恒定区(CL)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1、CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)和 免疫球蛋白轻链恒定区(CL)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1结构域、单结构域抗原结合位点(例如VHH)、免疫球蛋白CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白轻链可变结构域(VL)和免疫球蛋白CH1结构域；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、免疫球蛋白CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1结构域和单结构域抗原结合位点(例如VHH)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、免疫球蛋白CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)和免疫球蛋白CH1结构域；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、免疫球蛋白CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在一个实施方案中，本发明提供了这样的包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)和免疫球蛋白CH1结构域；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、单结构域抗原结合位点(例如VHH)、免疫球蛋白CH2、CH3和任选地CH4结构域。优选地，所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白。
在本发明提供的包含四条多肽链的抗体分子中，发明人还设计了能够稳定抗体分子结构和有利于各条链之间的正确偶合或配对的氨基酸残基。例如，在抗体分子的第二多肽链和第四多肽链的Fc结构域中包含具有“CPPC”氨基酸残基的铰链区，从而所述第二多肽链和第四多肽链彼此通过所述铰链区处氨基酸残基之间形成的二硫键稳定缔合。在一个实施方案中，本发明抗体分子的第二多肽链和第四多肽链在各自的Fc结构域中分别包含Y349C和S354C或者分别包含S354C和Y349C(根据Kabat等,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991)的EU索引进行编号，下文中称为“EU编号”)，由此，第二多肽链 和第四多肽链在Fc区进一步形成链间二硫键，以稳定第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明抗体分子的第二多肽链和/或第四多肽链在Fc结构域中包含影响抗体效应子功能的氨基酸突变。在一个具体实施方案中，所述效应子功能是抗体依赖的细胞介导的细胞毒性(ADCC)。在一个实施方案中，所述氨基酸突变存在于Fc区的CH2结构域，例如，所述抗体分子包含在第二多肽链和/或第四多肽链第234和235位置(EU编号)处的氨基酸置换。在一个具体实施方案中，所述氨基酸置换是L234A和L235A(下文中称为“LALA突变”)。
在一个实施方案中，本发明抗体分子的第二多肽链和第四多肽链各自的Fc结构域中分别包含凸起(“结(knob)”)或空穴(“扣(hole)”)，并且第二多肽链Fc结构域中的所述凸起或空穴可分别置于第四多肽链Fc结构域中的所述空穴或凸起中，由此所述第二多肽链和第四多肽链彼此形成“结入扣(knob-in-hole)”的稳定缔合。在一个实施方案中，在所述第二多肽链和第四多肽链之一条链中包含氨基酸置换T366W，并且在所述第二多肽链和第四多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(EU编号)。由此一条链中的凸起能够置于另一条链中的空穴中，促进第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明抗体分子的第一多肽链和第二多肽链的免疫球蛋白CL结构域和CH1结构域中分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于CL结构域中的所述空穴或凸起中，从而所述第一多肽链和第二多肽链彼此也形成“结入扣”的稳定缔合。同样地，本发明抗体分子的第三多肽链和第四多肽链的免疫球蛋白CL结构域和CH1结构域中也分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于CL结构域中的所述空穴或凸起中，从而所述第三多肽链和第四多肽链彼此也形成“结入扣”的稳定缔合。
在一个实施方案中，本发明抗体分子中的两个单结构域抗原结合位点结合第一抗原中的相同表位，两个Fab片段结合第二抗原中的相同表位，由此，所述抗体分子是针对第一抗原和第二抗原的双特异性抗体。
不特别地限制本发明的抗体分子特异性结合的抗原类型，抗原可以是例如细胞因子、生长因子、激素、信号传导蛋白、炎性介质、配体、细胞表面受体或其片段。在一个实施方案中，本发明的抗体分子特异性结合的抗原选自肿瘤相关抗原、免疫检查点分子、血管新生诱导因子、肿瘤坏死因子受体超家族成员和免疫系统中的共刺激分子，以及这些分子的配体和/或受体，例如，OX40、CD47、PD1、PD-L1、PD-L2、LAG-3、4-1BB(CD137)、VEGF和GITR。
本发明例示了如下所述的几种双特异性抗体。
i)在一个实施方案中，本发明的抗体分子是抗OX40/PD-L1双特异性抗体，所述抗体能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与表达在淋巴细胞表面的肿瘤坏死因子(TNF)受体家族成员OX40结合，由此活化T细胞，例如增强效应T细胞的免疫刺激/效应功能和/或使这些细胞增殖和/或下调调节性T细胞的免疫抑 制功能；并以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与肿瘤细胞表面的PD-L1结合，由此抑制T细胞上的PD-1与肿瘤细胞表面PD-L1的结合，诱导T细胞活化并发挥抗肿瘤作用。
在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体由左右基本上对称的4条多肽链组成，其中在左半部分的2条多肽链和右半部分的2条多肽链中，均包含(i)单结构域抗原结合位点；(ii)结合抗原的Fab片段；其中所述(i)位于所述(ii)的轻链可变结构域(VL)的N端或轻链恒定区(CL)的C端，或者所述(i)位于所述(ii)的重链可变结构域(VH)的N端或免疫球蛋白CH1结构域的C端，且所述(i)、(ii)分别结合OX40或PD-L1，所述(i)和(ii)之间具有或者不具有连接肽；以及位于所述(i)和(ii)的C端的(iii)免疫球蛋白Fc结构域。
在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体中的单结构域抗原结合位点是特异性结合PD-L1的VHH，Fab片段是特异性结合OX40的抗OX40抗体Fab片段。
在一个优选的实施方案中，本发明的抗OX40/PD-L1双特异性抗体中的所述特异性结合PD-L1的VHH包含SEQ ID NO:3所示的CDR1、SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；本发明的抗OX40/PD-L1双特异性抗体中的所述特异性结合OX40的抗OX40抗体Fab片段包含衍生自抗OX40抗体ADI-20112的SEQ ID NO:11/7所示的成对重链可变区序列/轻链可变区序列中的全部6个重链互补决定区(CDR)与轻链CDR，或者与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
又在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体中的所述特异性结合PD-L1的VHH包含SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列；本发明的抗OX40/PD-L1双特异性抗体中的所述特异性结合OX40的抗OX40抗体Fab片段包含衍生自抗OX40抗体ADI-20112的SEQ ID NO:11/7所示的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
又在一个优选的实施方案中，本发明的抗OX40/PD-L1双特异性抗体由左右基本上对称的4条多肽链组成，其中所述抗体分子的左半部分的2条多肽链分别包含SEQ ID NO:6所示的第一多肽链和SEQ ID NO:10所示的第二多肽链；分别包含SEQ ID NO:14所示的第一多肽链和SEQ ID NO:10所示的第二多肽链；分别包含SEQ ID NO:15所示的第一多肽链和SEQ ID NO:16所示的第二多肽链；分别包含SEQ ID NO:15所示的第一多肽链和SEQ ID NO:17所示的第二多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列；相应地，其中所 述抗体分子的右半部分的2条多肽链分别包含SEQ ID NO:6所示的第三多肽链和SEQ ID NO:10所示的第四多肽链；分别包含SEQ ID NO:14所示的第三多肽链和SEQ ID NO:10所示的第四多肽链；分别包含SEQ ID NO:15所示的第三多肽链和SEQ ID NO:16所示的第四多肽链；分别包含SEQ ID NO:15所示的第三多肽链和SEQ ID NO:17所示的第四多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
ii)在一个实施方案中，本发明的抗体分子是抗VEGF/GITR双特异性抗体，所述抗体能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与血管内皮生长因子(Vascular Endothelial Cell Growth Factor；VEGF)结合，从而阻断VEGF与其受体VEGFR的结合，使VEGFR无法活化，由此发挥抗血管生成的作用，例如，发挥抗肿瘤血管形成的作用，抑制肿瘤生长；并以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与CD4 +和CD8 +T细胞上的糖皮质激素诱导的肿瘤坏死因子受体(GITR)结合，由此逆转调节性T细胞(Treg)的抑制作用并共刺激和活化效应T细胞来发挥抗肿瘤作用。
在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体由左右基本上对称的4条多肽链组成，其中在左半部分的2条多肽链和右半部分的2条多肽链中，均包含(i)单结构域抗原结合位点；(ii)结合抗原的Fab片段；其中所述(i)位于所述(ii)的轻链可变结构域(VL)的N端或轻链恒定区(CL)的C端，或者所述(i)位于所述(ii)的重链可变结构域(VH)的N端或免疫球蛋白CH1结构域的C端，且所述(i)、(ii)分别结合VEGF或GITR，所述(i)和(ii)之间具有或者不具有连接肽；以及位于所述(i)和(ii)的C端的(iii)免疫球蛋白Fc结构域。
在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体中的单结构域抗原结合位点是特异性结合GITR的VHH，Fab片段是特异性结合VEGF的抗VEGF抗体Fab片段。
在一个优选的实施方案中，本发明的抗VEGF/GITR双特异性抗体中的所述特异性结合GITR的VHH包含GFAFGSS(SEQ ID NO:25)所示的CDR1、SGGGFGD(SEQ ID NO:26)所示的CDR2和ATDWRKP(SEQ ID NO:27)所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；本发明的抗VEGF/GITR双特异性抗体中的所述特异性结合VEGF的抗VEGF抗体Fab片段包含衍生自抗VEGF抗体Avastin的SEQ ID NO:22/20所示的成对重链可变区序列/轻链可变区序列中的全部6个重链互补决定区(CDR)与轻链CDR，或者与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
又在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体中的所述特异性结合GITR的VHH包含衍生自SEQ ID NO:24所示的抗GITR VHH氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列；本发明的抗VEGF/GITR双特异性抗体中的所述特异性结合VEGF的抗VEGF抗体Fab 片段包含衍生自抗VEGF抗体Avastin的SEQ ID NO:22/20所示的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
又在一个优选的实施方案中，本发明的抗VEGF/GITR双特异性抗体由左右基本上对称的4条多肽链组成，其中所述抗体分子的左半部分的2条多肽链分别包含SEQ ID NO:18所示的第一多肽链和SEQ ID NO:21所示的第二多肽链；分别包含SEQ ID NO:18所示的第一多肽链和SEQ ID NO:28所示的第二多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列；相应地，其中所述抗体分子的右半部分的2条多肽链分别包含SEQ ID NO:18所示的第三多肽链和SEQ ID NO:21所示的第四多肽链；分别包含SEQ ID NO:18所示的第三多肽链和SEQ ID NO:28所示的第四多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
在第二方面，本发明提供了编码本发明抗体分子中的任意一条或者多条多肽链的多核苷酸。
在第三方面，本发明提供了包含编码本发明抗体分子中的任意一条或者多条多肽链的多核苷酸的载体，优选地表达载体。
在第四方面，本发明提供了包含本发明多核苷酸或载体的宿主细胞。例如，所述宿主细胞是哺乳动物细胞，优选地是CHO细胞、HEK293细胞；所述宿主细胞是原核细胞，优选地是大肠杆菌细胞。
在第五方面，本发明提供了用于产生本发明抗体分子的方法，所述方法包括步骤(i)在适于表达所述抗体分子的条件下培养本发明的宿主细胞，和(ii)从所述宿主细胞或所述培养基回收所述抗体分子。
在第六方面，本发明提供了包含本发明抗体分子的免疫缀合物和药物组合物。本文公开的抗体分子可以单独或与其他药物或其他治疗模式联合用来治疗、预防和/或诊断疾病，如自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)、肿瘤等。
在第七方面，本发明提供了本发明的抗体分子、免疫缀合物和药物组合物的用途，用作在个体中治疗和/或预防疾病的药物或用作疾病的诊断工具。优选地，所述个体是哺乳动物，更优选地是人。在一个实施方案中，所述疾病是自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)、肿瘤。
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1A－1D例示了本发明双特异性抗体的4种结构。
图2A－2D分别显示了利用大小排阻层析(size exclusion chromatography；SEC)检测的本发明制备的4种结构的抗OX40/PD-L1双特异性抗体Bi-110-112HC、Bi-113-112HC、Bi-119-112LC和Bi-122-112LC的纯度。
图3显示了通过FACS检测的抗OX40/PD-L1双特异性抗体Bi-119-112LC、以及作为对照的抗PD-L1人源化Nb-Fc抗体与过量表达PD-L1的CHO细胞的结合。图中横轴表示抗体浓度、纵轴表示平均荧光强度(MFI)。
图4显示了通过FACS检测的抗OX40/PD-L1双特异性抗体Bi-119-112LC、以及作为阳性对照的抗OX40抗体ADI-20112与过量表达OX40的CHO细胞的结合。图中横轴表示抗体浓度、纵轴表示平均荧光强度(MFI)。
图5显示了抗OX40/PD-L1双特异性抗体对过量表达OX40的CHO细胞和过量表达PD-L1的CHO细胞的同时结合作用。
图6显示了本发明的抗OX40/PD-L1双特异性抗体对人PD-1与PD-L1结合的作用，证明本发明的双特异性抗体Bi-119-112LC阻断人PD-1与PD-L1的结合。还检测了作为对照的抗PD-L1人源化Nb-Fc和IgG1的作用。
图7显示了本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC有效解除PD1/PD-L1相互作用对NFAT信号通路的阻断效应，进而获得了荧光信号。还检测了作为对照的抗PD-L1人源化Nb-Fc和IgG1的作用。
图8显示了本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC对PD-L1依赖的OX40介导的信号通路的作用。还检测了抗PD-L1人源化Nb-Fc、ADI-20112、抗PD-L1人源化Nb-Fc+ADI-20112和IgG1的作用。
图9显示了差示扫描荧光法(DSF)测定的本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC的T m值的结果。
图10显示了本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC对人T细胞的激活作用。还检测了抗PD-L1人源化Nb-Fc、ADI-20112和IgG1的作用。
图11A－11B例示了本发明双特异性抗体的2种结构。
图12A－12B分别显示了利用SEC检测的本发明制备的抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51的纯度。
图13显示了通过FACS检测的抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51与过量表达GITR的CHO细胞的结合。图中横轴表示抗体浓度、纵轴表示平均荧光强度(MFI)。
发明详述
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
术语“抗体”在本文中以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体和抗体片段。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指天然存在的包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
术语“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv、双体抗体(diabody)、单结构域抗体(sdAb)。所述Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab') 2，其为Fab’的二聚体，是二价的抗体片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，由此将F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段(其它抗体片段的更详细的描述请参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993))。所述Fv片段由抗体单臂的VL和VH结构域组成。另外，虽然Fv片段的两个结构域VL和VH由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性连接肽连接，在所述单条蛋白链中VL区和VH区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗体片段。
术语“单结构域抗体”(sdAb)或“单可变结构域(SVD)抗体”通常指这样的抗体，其中单个可变结构域(例如，重链可变结构域(VH)或轻链可变结构域(VL)、衍生自骆驼科重链抗体的重链可变结构域、衍生自鱼类IgNAR的VH样单结构域(v-NAR))即可赋予抗原结合。即，该单个可变结构域不需要与另一可变结构域相互作用以识别靶抗原。单结构域抗体的实例包括源自骆驼科(美洲驼和骆驼)和软骨鱼(例如护士鲨)的单结构域抗体(WO 2005/035572)。
术语“骆驼化的人VH结构域”是指将衍生自骆驼科VHH的关键元件转移到人VH结构域上导致人VH结构域不再需要与VL结构域配对来识别靶抗原，经骆驼化的人VH结构域单独即可赋予抗原结合特异性。
如本文所用的术语“结合位点”或“抗原结合位点”表示抗体分子中与抗原实际结合的区域，包括由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)组成的VH/VL对、衍生自骆驼科重链抗体的重链可变结构域、来自鲨鱼科动物的IgNAR的VH样单结构域(v-NAR)、骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域。在本发明的一个实施方案中，本发明的抗体分子包含至少四个抗原结合位点，例如，包含两个单结构域抗原结合位点(例如，VHH)和两个Fab片段中VH/VL对形成的抗原结合位点。
术语“单结构域抗原结合位点”表示抗体分子的以单个可变结构域(例如，重链可变结构域(VH)、轻链可变结构域(VL)、衍生自骆驼科重链抗体的重链可变结构域、来自鲨鱼科动物的IgNAR的v-NAR、骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域、和它们的经重组的单结构域)与抗原实际结合的区域。在本发明的一个实施方案中，本发明的抗体分子包含两个单结构域抗原结合位点，分别结合相同或不同的抗原。在本发明的另一个实施方案中，本发明的抗体分子包含两个单结构域抗原结合位点，分别结合相同或不同的抗原表位。
如本文所用，术语“单特异性”抗体指具有一个或多个结合位点的抗体，所述位点中的每一个位点与相同抗原的相同表位结合。
如本文所用，术语“多特异性”抗体指具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。本文提供的抗体通常是多特异性抗体，例如双特异性抗体。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。在一个实施方案中，本文提供了这样的双特异性抗体，其具有针对第一抗原和第二抗原的结合特异性。
术语“免疫球蛋白分子”指具有天然存在抗体的结构的蛋白质。例如，IgG类免疫球蛋白是由二硫键键合的两条轻链和两条重链组成的约150,000道尔顿的异四聚体糖蛋白。从N端至C端，每条免疫球蛋白重链具有一个重链可变区(VH)，也称作重链可变结构域，随后是三个重链恒定结构域(CH1、CH2和CH3)。类似地，从N端至C端，每条免疫球蛋白轻链具有一个轻链可变区(VL)，也称作轻链可变结构域，随后是一个轻链恒定结构域(CL)。免疫球蛋白的重链可以归属5个类别之一，称作α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，其中某些类别可以进一步划分成亚类，例如γ 1(IgG1)、γ 2(IgG2)、γ 3(IgG 3)、γ 4(IgG 4)、α 1(IgA 1)和α 2(IgA 2)。免疫球蛋白的轻链可以基于其恒定结构域的氨基酸序列而划分成两种类型之一，称作κ和λ。免疫球蛋白基本上由借助免疫球蛋白铰链区连接的两个Fab分子和一个Fc结构域组成。
术语“Fc结构域”或“Fc区”在本文中用来定义免疫球蛋白重链的含有至少一部分恒定区的C端区域。该术语包括天然序列Fc区和变体Fc区。天然的免疫球蛋白“Fc结构域”包含两个或三个恒定结构域，即CH2结构域、CH3结构域和可选的CH4结构域。例如， 在天然抗体中，免疫球蛋白Fc结构域包含源自IgG、IgA和IgD类抗体的两条重链的第二和第三恒定结构域(CH2结构域和CH3结构域)；或者包含源自IgM和IgE类抗体的两条重链的第二、第三和第四恒定结构域(CH2结构域、CH3结构域和CH4结构域)。除非本文中另外说明，否则Fc区或重链恒定区中的氨基酸残基编号根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes of Health,Bethesda,MD,1991中所述的EU编号体系(也称作EU索引)进行编号。
术语“效应子功能”指随免疫球蛋白同种型变动的归因于免疫球蛋白Fc区的那些生物学活性。免疫球蛋白效应子功能的例子包括：C1q结合和补体依赖的细胞毒性(CDC)、Fc受体结合作用、抗体依赖的细胞介导的细胞毒性(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞摄取抗原、下调细胞表面受体(例如B细胞受体)和B细胞活化。
术语“嵌合抗体”是这样的抗体分子，其中(a)将恒定区或其部分改变、替换或交换，从而抗原结合位点与不同的或改变的类别、效应子功能和/或物种的恒定区或赋予嵌合抗体新性能的完全不同的分子(例如，酶、毒素、激素、生长因子、药物)等连接；或(b)将可变区或其部分用具有不同或改变的抗原特异性的可变区改变、替换或交换。例如，小鼠抗体可以通过将其恒定区更换为来自人免疫球蛋白的恒定区进行修饰。由于更换为人类恒定区，该嵌合抗体可以保留其在识别抗原方面的特异性，同时如与原始小鼠抗体相比，具有在人类中降低的抗原性。
“人源化抗体”是一种保留非人类抗体(例如小鼠单克隆抗体)的抗原特异性反应性，同时作为治疗药对人施用时免疫原性较低的抗体。这可以例如通过保留非人类抗原结合位点并且抗体的剩余部分替换成它们的人类相应部分(即，恒定区以及可变区中不参与结合的部分为人类抗体的相应部分)来实现。参见，例如Padlan,Anatomy of the antibody molecule，Mol.Immun.,1994，31:169-217。人类抗体工程化技术的其他例子包括但不限于US 5,766,886中公开的Xoma技术。
术语“…价”抗体指抗体分子中存在的抗原结合位点的数目。“二价”、“三价”和“四价”抗体指抗体分子中分别存在2个抗原结合位点、3个结合位点和4个结合位点。在一个实施方案中，本文中报道的双特异性抗体是“四价的”。
术语“由左右基本上对称的4条多肽链组成”的抗体是指抗体分子由4条多肽链组成，包括抗体分子左边的2条多肽链和右边的2条多肽链，且抗体分子左边的2条多肽链的序列和右边的2条多肽链的序列具有100％同一性或至少95％或至少99％同一性。
术语“柔性连接肽”或“连接肽”是指由氨基酸组成的连接肽，例如单独或组合使用的甘氨酸和/或丝氨酸残基，以连接抗体中的各个可变结构域。在一个实施方案中，柔性连接肽是Gly/Ser连接肽，包括氨基酸序列(Gly 4Ser)n，其中n是等于或大于1的正整数，例如，n是1－7中的正整数。在一个实施方案中，所述柔性连接肽是(Gly 4Ser) 2(SEQ ID NO:9)。还包括在本发明范围内的是WO2012/138475中描述的连接肽，其通过引用并入 本文。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗原结合位点与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法测定。
“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由解离常数(K D)代表，解离常数是解离速率常数和缔合速率常数(分别是k dis和k on)的比例。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。在本文的一些实施方案中，第一抗原、第二抗原、第三抗原是三种不同的抗原。
术语“肿瘤相关抗原”或“癌症抗原”可互换地指与正常细胞相比，优选在癌细胞表面完全或作为片段(例如，MHC/肽)表达的分子(通常为蛋白质、碳水化合物或脂质)，并且所述分子可用在药剂对癌细胞的优先靶向中。在一些实施方案中，肿瘤相关抗原是与正常细胞相比在肿瘤细胞中过表达的细胞表面分子，例如与正常细胞相比1倍过量表达、2倍过量表达、3倍过量表达或更多倍过量表达。在一些实施方案中，肿瘤相关抗原是在肿瘤细胞中不适当地合成的细胞表面分子，例如与正常细胞上表达的分子相比含有缺失、添加或突变的分子。在一些实施方案中，肿瘤相关抗原仅在肿瘤细胞的细胞表面完整表达或作为片段表达，并且不在正常细胞的表面上合成或表达。现有技术中公开了诸多肿瘤相关抗原，例如，表皮生长因子受体变体III(EGFRvIII)、肿瘤相关的糖蛋白72(TAG72)、癌胚抗原(CEA)、上皮细胞粘附分子(EPCAM)、白介素11受体α(IL-11Ra)、血管内皮生长因子受体2(VEGFR2)、表皮生长因子受体(EGFR)、神经细胞粘附分子(NCAM)、胰岛素样生长因子1受体(IGF-I受体)、黑素瘤相关抗原1(MAGE-A1)、CD72、CD47等。
术语“免疫检查点”意指免疫系统中存在的一类抑制性信号分子，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受(Pardoll DM.,The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer,2012,12(4):252-264)。研究发现，肿瘤细胞能够逃避体内免疫系统而失控增殖的原因之一是利用了免疫检查点的抑制性信号通路，由此抑制了T淋巴细胞活性，使得T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S,Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov,2013,12(2):130-146)。免疫检查点分子包括但不限于程序性死亡1(PD-1)、PD-L1、PD-L2、细胞毒T淋巴细胞抗原4(CTLA-4)、LAG-3和TIM-3。
术语“共刺激分子”是指T细胞上的与共刺激配体特异性结合从而介导T细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受 体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是OX40、GITR、4-1BB(即CD137)、CD27和/或CD28。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本文中使用的，术语细胞因子包括来自天然来源或来自重组细胞培养物的蛋白质及天然序列细胞因子的生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。
“免疫缀合物”是与一个或多个异源分子(包括但不限于细胞毒性剂)缀合的抗体。
如本文所用，术语“细胞毒性剂”指抑制或阻止细胞功能和/或造成细胞死亡或破坏的物质。细胞毒性剂包括但不限于放射性同位素(例如，At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32、Pb 212和Lu的放射性同位素)；化疗药或药物(例如，甲氨蝶呤、阿霉素、长春碱类生物碱(长春新碱、长春碱、依托泊苷)、多柔比星、美法仑、丝裂霉素C、苯丁酸氮芥、佐柔比星或其他嵌入剂)；生长抑制剂；酶及其片段如溶核酶；抗生素；毒素如细菌源、真菌源、植物源或动物源的小分子毒素或酶活性毒素，包括其片段和/或变体；和下文公开的各种抗肿瘤剂或抗癌剂。
氨基酸序列的“同一性百分数(％)”是指将候选序列与本说明书中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。
对于多肽序列，“保守性修饰”包括对多肽序列的置换、缺失或添加，它们导致某个氨基酸置换为化学上相似的氨基酸。提供功能上相似氨基酸的保守性置换表是本领域熟知的。这类保守性修饰的变体相对于本发明的多态性变体、物种间同源物和等位基因而言是附加的并且不排斥它们。以下8组含有互为保守替换的氨基酸：1)丙氨酸(A)、甘氨酸(G)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；7)丝氨酸(S)、苏氨酸(T)；和8)半胱氨酸(C)、甲硫氨酸(M)(参阅例如，Creighton,Proteins(1984))。在一些实施方案中，术语“保守序列修饰”用于指不显著影响或改变含有氨基酸序列的抗体的结合特征的氨基酸修饰。
术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞，包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，这包括原代转化的细胞和从其衍生的子代。宿主细胞是可以用来产生本发明抗体分子的任何类型的细胞系统，包括真核细胞，例如，哺乳动物细胞、昆虫细胞、酵母细胞；和原核细胞，例如，大肠杆菌细胞。宿主细胞包括培 养的细胞，也包括转基因动物、转基因植物或培养的植物组织或动物组织内部的细胞。
术语“表达载体”是指包含重组多核苷酸的载体，其包含有效连接要表达的核苷酸序列的表达控制序列。表达载体包含足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞提供或在体外表达系统中。表达载体包括本领域已知的所有那些，包括被掺入重组多核苷酸的粘粒、质粒(例如，裸的或包含在脂质体中)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。
术语“个体”或“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体是人。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体分子用来延缓疾病发展或用来减慢疾病的进展。
术语“抗肿瘤作用”指可以通过多种手段展示的生物学效果，包括但不限于例如，肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃癌(包括胃肠癌和胃肠基质癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，尿道癌，肝瘤，乳腺癌，结肠癌，直肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌，肛门癌，阴茎癌，黑素瘤，浅表扩散性黑素瘤，恶性雀斑样痣黑素瘤，肢端黑素瘤，结节性黑素瘤，多发性骨髓瘤和B细胞淋巴瘤，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖，脑瘤和脑癌，以及头颈癌，及相关转移。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括肺癌(例如非小细胞肺癌)、肝癌、胃癌或结肠癌，包括那些癌症的转移性形式。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”、“癌性”和“肿瘤”在本文中提到时并不互相排斥。
术语“感染性疾病”是指病原体引发的疾病，包括例如病毒感染、细菌感染、寄生虫感染或真菌感染。
II.本发明的抗体分子
本发明提供了一种新型的抗体分子，其能够用于多种疾病的免疫治疗、预防和/或诊断。本发明的抗体分子至少包含4个抗原结合位点，其能够作为单特异性抗体或多特异性(例如双特异性)抗体发挥作用，优选地，其能够作为多特异性(例如双特异性)抗体发挥作用。
在产生具有多条多肽链的单特异性或多特异性(例如双特异性)抗体时，通常会发生不希望的链间错配、抗体亲和力降低、稳定性降低等问题。本申请构建的抗体分子能够避免这些常见问题。
本申请所构建的抗体分子平台包含(i)单结构域抗原结合位点；(ii)结合抗原的Fab片段；其中所述(i)位于所述(ii)的轻链可变结构域(VL)的N端或轻链恒定区(CL)的C端，或者所述(i)位于所述(ii)的重链可变结构域(VH)的N端或免疫球蛋白CH1结构域的C端，且所述(i)和(ii)分别结合相同或者不同的抗原，所述(i)和(ii)之间具有或者不具有连接肽；以及位于所述(i)和(ii)的C端的(iii)免疫球蛋白Fc结构域。
在一个实施方案中，本发明抗体分子具有4条多肽链，其包含2个单结构域抗原结合位点和2个Fab片段以及Fc区。
在另一个实施方案中，本发明抗体分子的单结构域抗原结合位点和Fab片段之间不具有连接肽。
又在一个实施方案中，本发明抗体分子的单结构域抗原结合位点和Fab片段之间具有连接肽。不特别地限制所述连接肽的类型。在实施方案中，所述连接肽是具有1至100个、特别地1至50个、更特别地1至20个氨基酸长度的氨基酸序列的肽。在一些实施方案中，所述肽连接肽是(GxS)n或(GxS)nGm，其中G＝甘氨酸，S＝丝氨酸并且x＝1－4中的任一整数，n＝1－7中的任一整数，以及m＝0－3中的任一整数。在一个具体实施方案中，所述连接肽是(G 4S) 2(SEQ ID NO：9)。
本发明抗体分子中的单结构域抗原结合位点是能够以较高亲和力特异性结合靶抗原表位的单个可变结构域，例如，重链可变结构域(VH)、轻链可变结构域(VL)、衍生自骆驼科重链抗体的重链可变结构域、来自鲨鱼科动物的IgNAR的v-NAR、骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域、和它们的经重组的单结构域。在一个实施方案中，本发明抗体分子中的单结构域抗原结合位点是衍生自骆驼科重链抗体的重链可变结构域、骆驼化的人VH结构域和/或人源化的骆驼科抗体重链可变结构域。
现有技术中已经对从骆驼科物种(例如骆驼、羊驼、单峰驼、驼羊和原驼)获得的抗体蛋白的大小、结构和针对人类受试者的抗原性进行了表征。在自然界中来自骆驼科哺乳动物家族的某些IgG抗体缺少轻链，并且因此在结构上区别于来自其他动物的具有两条重链和两条轻链的常见四链抗体结构。参见PCT/EP 93/02214(1994年3月3日公布的WO 94/04678)。
可以通过基因工程方法获得骆驼科重链抗体的对靶抗原具有高亲和力的重链可变结构域(该区域也称为VHH)。参见1998年6月2日授予的美国专利号5,759,808。与其他非人源抗体片段一样，骆驼科VHH的氨基酸序列可以重组地改变以获得更逼真模仿人序列 的序列，即，“人源化”，由此降低骆驼科VHH对人类的抗原性。另外，也可以将衍生自骆驼科VHH的关键元件转移到人VH结构域上，获得骆驼化的人VH结构域。在本发明的一个实施方案中，本发明抗体分子中的单结构域抗原结合位点是针对PD-L1的人源化VHH，其具有SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列。在本发明的另一个实施方案中，本发明抗体分子中的单结构域抗原结合位点是针对GITR的VHH，其具有SEQ ID NO:24所示的氨基酸序列。
VHH的分子量是人IgG分子的分子量的十分之一，并且具有仅数纳米的物理直径。VHH本身具有极高的热稳定性、对极端pH和蛋白酶解消化稳定和抗原性低，因此，在本发明抗体分子的一个实施方案中，包含VHH作为构建模块对本发明抗体分子的稳定性、对人受试者的低抗原性做出了贡献。
本发明抗体分子中的Fab片段能够以较高亲和力特异性结合靶抗原表位。在一个实施方案中，所述Fab片段是免疫球蛋白的Fab片段，其包含由免疫球蛋白轻链可变区(VL)和免疫球蛋白轻链恒定区(CL)组成的肽；且包含由免疫球蛋白重链可变区(VH)和免疫球蛋白重链恒定区1(CH1)组成的肽；其中所述CL区和CH1区任选地通过二硫键共价连接而异源二聚化Fab片段。在一个实施方案中，所述Fab片段是免疫球蛋白Fab片段的轻链可变区(VL)和轻链可变区(VL)交换后的Fab片段，其包含由免疫球蛋白轻链可变区(VL)和重链恒定区(CH1)组成的肽；且包含由免疫球蛋白重链可变区(VH)和轻链恒定区(CL)组成的肽；其中所述CL区和CH1区任选地通过二硫键共价连接而异源二聚化Fab片段。又在一个实施方案中，所述Fab片段是免疫球蛋白Fab片段的轻链恒定区(CL)和重链恒定区(CH1)交换后的Fab片段，其包含由免疫球蛋白重链可变区(VH)和轻链恒定区(CL)组成的肽；且包含由免疫球蛋白轻链可变区(VL)和重链恒定区(CH1)组成的肽；其中所述CL区和CH1区任选地通过二硫键共价连接而异源二聚化Fab片段。
如本领域技术人员所认识到的，Fab片段的CL区和CH1区之间的二硫键是优选的，但对于功能不是必需的( Orcutt KD等人，A modular IgG-scFv bispecific antibody topology， Protein Eng Des Sel.2010，23(4):221-228)。因此，在一些实施方案中，本发明抗体分子中的Fab片段不包含二硫键。在这方面，Fab片段的两条链可以以这样的方式进行改造，从而在无需二硫键的情况下稳定地相互作用。例如，在一些实施方案中，可改造Fab片段的两条链以去除半胱氨酸残基，并且Fab片段的两条链仍然稳定地相互作用并如Fab一样发挥功能。在一个实施方案中，对Fab片段的两条链进行突变以促进这两条链之间稳定的相互作用。例如，可用“结入扣”遗传改造策略(参见例如John B.B.Ridgway等人，‘Knobs-into-holes’engineering of antibody CH3domains for heavy chain heterodimerization.Protein Engineering,1996.9(7):p.617-21；Shane Atwell等人，Stable heterodimers form remodeling the domain interface of a homodimer using a phage display library.J.Mol.Biol,1997.270:p.26-35)来促进Fab片段的两条链之间的异二聚化。利用该策略，通过在相互作用结构域之间的界面处用较大的氨基酸侧链取代小的氨基酸侧链来产生“结”结构。相应地，通过在相互作用分子之间的界面处用小的侧链替代大的侧链 来实现“扣”结构。因此，还考虑用于本文的是出于特定目的而设计变异Fab片段，例如在CH1和/或CL的恒定结构域中的氨基酸改变，以及二硫键的去除等。
在一些实施方案中，本发明抗体分子中的Fab片段来源于单克隆抗体并且可来源于任何类型的抗体，包括IgA、IgM、IgD、IgG、IgE及其亚型，例如IgG1、IgG2、IgG3和IgG4。轻链结构域可来源于κ链或λ链。另外，本文所用的Fab片段也可通过重组制备。在一些实施方案中，本发明抗体分子的Fab片段中的CH1结构域、CL结构域均来自人免疫球蛋白的相应部分或具有与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
本发明抗体分子中的免疫球蛋白Fc结构域能够延长本发明抗体的体内半寿期和提供效应子功能。参见，例如，国际公开号WO98/23289；国际公开号WO97/34631；和美国专利号6 277 375。
在一个具体实施方案中，在本发明抗体分子的第二多肽链和第四多肽链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，由此，本发明抗体分子的第二多肽链和第四多肽链在Fc区形成链间二硫键，这也促成了本发明的抗体分子的第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明的抗体分子的免疫球蛋白Fc结构域也使用了“结入扣”技术，该技术可在本发明抗体分子的不同链之间改造界面，以促进本发明抗体分子的各条链正确缔合。通常，该技术涉及在一条链的界面引入“凸起”，在欲与之配对的另一条链的界面引入相应的“空穴”，使得凸起可置于空穴中。第一个优选的界面包含一条链的重链恒定结构域的CH3结构域和欲与之配对的另一条链的重链恒定结构域的CH3结构域。可通过将来自一条链的重链恒定结构域的CH3结构域的界面的小氨基酸侧链替换为较大的侧链(例如酪氨酸或色氨酸)来构建凸起。通过将大氨基酸侧链替换为较小的侧链(例如丙氨酸或苏氨酸)，在欲配对的另一条链的重链恒定结构域的CH3结构域的界面构建与凸起相同或相似大小的补偿性空穴。第二个优选的界面是上文所述Fab片段的包含轻链的CL结构域和重链的CH1结构域，通过构建凸起-空穴相互作用促进Fab片段的两条链之间发生正确的异二聚化。
在一个实施方案中，本发明抗体分子的Fc区包含对Fc受体的结合亲和力的修饰。在一个实施方案中，所述Fc受体是Fcγ受体，特别地是人Fcγ受体。在一个实施方案中，所述Fc受体是活化性Fc受体。在一个实施方案中，所述修饰减少本发明抗体分子的的效应子功能。在一个具体实施方案中，所述效应子功能是抗体依赖的细胞介导的细胞毒性(ADCC)。在一个实施方案中，所述修饰在所述免疫球蛋白分子Fc区内，特别地在其CH2区内。在一个实施方案中，所述免疫球蛋白分子包含在免疫球蛋白重链第329位置(EU编号)处的氨基酸置换。在一个具体实施方案中，所述氨基酸置换是P329G。在一个实施方案中，本发明抗体分子包含在免疫球蛋白重链第234和235位置(EU编号)处的氨基酸置换。在一个具体实施方案中，所述氨基酸置换是L234A和L235A(LALA突变)(Armour KL等人，Recombinant human IgG molecules lacking Fcgamma receptor I binding  and monocyte triggering activities.Eur J Immunol,1999.29(8):2613-24)。在一个实施方案中，本发明抗体分子包含在免疫球蛋白重链第234、235和329位置处(EU编号)的氨基酸置换。在一个具体实施方案中，所述免疫球蛋白分子包含免疫球蛋白重链中的氨基酸置换L234A、L235A和P329G(EU编号)。
本发明抗体分子中的至少一个单结构域抗原结合位点(例如，两个单结构域抗原结合位点)和至少一个Fab片段能够特异地结合至少一种抗原。优选地，本发明的抗体分子结合两种或两种以上的抗原，由此，本发明的抗体分子是多特异性抗体分子，例如，双特异性抗体分子。所述抗原包括但不限于细胞因子、生长因子、激素、信号传导蛋白、炎性介质、配体、细胞表面受体或其片段。
在一些实施方案中，本发明的抗体分子抑制多种(例如两种)免疫检查点分子的信号传导通路，例如，本发明的抗体分子是具有针对PD-L1的第一结合特异性和针对TIM-3、LAG-3、PD-1或PD-L2的第二结合特异性的双特异性抗体分子，通过抑制所述免疫检查点分子的信号传导通路发挥作用。
在一些实施方案中，本发明的抗体分子抑制免疫检查点分子的信号传导通路和激动共刺激分子的信号传导通路，例如，本发明的抗体分子是具有针对PD-L1、TIM-3、LAG-3、PD-1或PD-L2的第一结合特异性和针对OX40、GITR、4-1BB、CD27或CD28的第二结合特异性的双特异性抗体分子，通过抑制所述免疫检查点分子的信号传导通路和通过激动所述共刺激分子的信号传导通路而发挥作用。
在一些实施方案中，本发明的抗体分子抑制免疫检查点分子的信号传导通路和抑制异常血管生成，例如，本发明的抗体分子是具有针对PD-L1、TIM-3、LAG-3、PD-1或PD-L2的第一结合特异性和针对VEGF或VEGF受体的第二结合特异性的双特异性抗体分子，通过抑制所述免疫检查点分子的信号传导通路和通过抑制VEGF、VEGF受体的信号传导通路而发挥作用。
在一些实施方案中，本发明的抗体分子激动多种(例如两种)共刺激分子的信号传导通路，例如，本发明的抗体分子是具有针对OX40的第一结合特异性和针对GITR、4-1BB、CD27或CD28的第二结合特异性的双特异性抗体分子，通过激动所述共刺激分子的信号传导通路发挥作用。
在一些实施方案中，本发明的抗体分子激动共刺激分子的信号传导通路和抑制异常血管生成，例如，本发明的抗体分子是具有针对OX40、GITR、4-1BB、CD27或CD28的第一结合特异性和针对VEGF或VEGF受体的第二结合特异性的双特异性抗体分子，通过激动所述共刺激分子的信号传导通路和通过抑制VEGF、VEGF受体的信号传导通路而发挥作用。
在一些实施方案中，本发明的抗体分子抑制异常血管生成、抑制免疫检查点分子的信号传导通路和激动共刺激分子的信号传导通路，例如，本发明的抗体分子是具有针对VEGF或VEGF受体的第一结合特异性、针对PD-L1、TIM-3、LAG-3、PD-1或PD-L2的第二结合特异性和针对OX40、GITR、4-1BB、CD27或CD28的第三结合特异性的三 特异性抗体分子，通过抑制VEGF、VEGF受体的信号传导通路、抑制所述免疫检查点分子的信号传导通路和通过激动所述共刺激分子的信号传导通路而发挥作用。
在一些实施方案中，本发明的抗体分子具有说明书附图1A－1D中例示的任一结构。
如图1A中的示意图所示，本发明的示例性抗体分子是四链抗体分子，包含2个Fab片段、分别位于每一Fab片段轻链恒定区(CL)C端的单结构域抗原结合位点、以及作为本发明抗体分子C端的免疫球蛋白Fc结构域，其中在Fab片段轻链恒定区(CL)C端和单结构域抗原结合位点之间具有或者不具有连接肽。
如图1B中的示意图所示，本发明的示例性抗体分子是四链抗体分子，包含2个Fab片段、分别位于每一Fab片段轻链可变结构域(VL)N端的单结构域抗原结合位点、以及作为本发明抗体分子C端的免疫球蛋白Fc结构域，其中在Fab片段轻链可变结构域(VL)N端和单结构域抗原结合位点之间具有或者不具有连接肽。
如图1C中的示意图所示，本发明的示例性抗体分子是四链抗体分子，包含2个Fab片段、分别位于每一Fab片段CH1结构域C端的单结构域抗原结合位点、以及作为本发明抗体分子C端的免疫球蛋白Fc结构域，其中在Fab片段CH1结构域C端和单结构域抗原结合位点之间具有或者不具有连接肽。
如图1D中的示意图所示，本发明的示例性抗体分子是四链抗体分子，包含2个Fab片段、分别位于每一Fab片段重链可变结构域(VH)N端的单结构域抗原结合位点、以及作为本发明抗体分子C端的免疫球蛋白Fc结构域，其中在Fab片段重链可变结构域(VH)N端和单结构域抗原结合位点之间具有或者不具有连接肽。
下面例示一些本发明的抗体分子和本发明的抗体分子对其特异性结合的抗原所参与的信号传导通路的调节作用。
i)在一个实施方案中，本发明的抗体分子是抗OX40/PD-L1双特异性抗体或多特异性抗体。
OX40(也称为CD134、TNFRSF4和ACT35)是细胞表面糖蛋白和肿瘤坏死因子(TNF)受体超家族成员，在T淋巴细胞上表达并且为活化的T细胞的增殖和存活提供共刺激信号。OX40最初被描述为大鼠CD4T细胞上的T细胞激活标志物(Paterson DJ等人，Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts.Mol Immunol.1987；24:1281–1290)并且随后显示为在TCR招募中被上调(Mallett S.等人，Characterization of the MRC OX40 antigen of activated CD4positive T lymphocytes--a molecule related to nerve growth factor receptor.EMBO J.1990；9:1063–1068)。已在CD4+T细胞、CD8+T细胞、NK细胞、NKT细胞和嗜中性粒细胞上鉴定到OX40(Paterson D.J.等人，Antigens of activated Rat T lymphocytes including a molecule of 50,000 M(r)detected only on CD4 positive T blasts,Molecular Immunology,1987，24(12):1281–1290)。OX40信号传导能够促进对T细胞的共刺激信号，导致增强的细胞增殖、存活、效应子功能和迁移(Gramaglia I等人，Ox-40ligand:a potent costimulatory molecule for sustaining primary CD4T cell responses.J Immunol.1998； 161:6510–6517；Gramaglia I等人，The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion.J Immunol.2000；165:3043–3050)。
现有技术中公开了作为OX40激动剂的抗OX40抗体。例如，WO 2012/027328中公开了抗OX40抗体mAb 106-222和人源化106-222(Hu106)的重链可变区和轻链可变区的氨基酸序列；抗OX40抗体mAb 119-122和人源化119-122(Hu119)的重链可变区和轻链可变区的氨基酸序列。美国专利号7,959,925、PCT公开号WO 2006/121810和中国专利申请号201710185399.9中也公开了作为OX40激动剂的抗OX40抗体。所述抗OX40抗体能够活化OX40，从而诱导效应T淋巴细胞增殖，促进针对表达肿瘤相关抗原(TAA)的肿瘤细胞的免疫应答。
PD-L1(也称作分化抗原簇274(CD274)或B7同源物1(B7-H1))，是40kDa I型跨膜蛋白。PD-L1与活化的T细胞上存在的其受体PD-1结合，下调T细胞活化(Latchman等人，2001 Nat Immunol 2：261-8；Carter等人，2002 Eur J Immunol 32：634-43)。已经在许多癌中发现了PD-L1表达，包括人肺癌、卵巢癌、结肠癌和多种骨髓瘤等，并且PD-L1表达经常与癌的预后不良相关(Iwai等人(2002)PNAS 99:12293-7；Ohigashi等人(2005)Clin Cancer Res 11:2947-53；Okazaki等人(2007)Intern.Immun.19:813-24；Thompson等人(2006)Cancer Res.66:3381-5)。已经提出通过抑制PD1与PD-L1的局部相互作用可以在一部分肿瘤患者中逆转免疫抑制。
罗氏(Roche)研发的抗PD-L1抗体Atezolizumab、德国默克(Merck KGaA)和美国辉瑞(Pfizer)合作开发的抗PD-L1抗体Avelumab、阿斯利康研发的Durvalumab显示了对部分肿瘤患者的治疗效果。其它抗PD-L1抗体包括YW243.55.S70(重链和轻链可变区显示在WO2010/077634中的SEQ ID NOs 20和21中)和WO2007/005874中公开的抗PD-L1抗体等。
本发明的抗OX40/PD-L1双特异性抗体或多特异性抗体至少同时靶向OX40和PD-L1，其Fab片段和单结构域抗原结合位点分别结合OX40或PD-L1分子，能够阻断抑制性PD-1/PD-L1信号传导途径且活化T细胞和自然杀伤(NK)细胞中的OX40/OX40配体信号传导途径，促进针对疾病的免疫应答。
在一个实施方案中，本发明的抗体分子包含特异性结合PD-L1的单结构域抗原结合位点和特异性结合OX40的Fab片段。在一个实施方案中，本发明的抗体分子包含特异性结合OX40的单结构域抗原结合位点和特异性结合PD-L1的Fab片段。
对于所述特异性结合PD-L1或OX40的Fab片段，其包含衍生自任何现有技术中报导的抗PD-L1抗体(例如，上文中例举的抗PD-L1抗体)和将来研发出的抗PD-L1抗体VH/VL对的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；或者包含衍生自任何现有技术中报导的抗OX40抗体(例如，上文中例举的抗OX40抗体)和将来研发出的抗OX40抗体VH/VL对的6个CDR或与所述6个CDR中的一个或多个CDR 具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。在一个实施方案中，抗OX40抗体是ADI-20112，其具有SEQ ID NO:10所示的重链氨基酸序列和SEQ ID NO:15所示的轻链氨基酸序列。
对于所述特异性结合PD-L1或OX40的单结构域抗原结合位点，其包含特异性结合PD-L1或OX40的重链可变结构域(VH)、轻链可变结构域(VL)、来自骆驼科血清的天然不含轻链的仅由两条重链组成的骆驼抗体中的重链可变结构域、来自鲨鱼科动物的IgNAR的VH样单结构域、骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域。
在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体包含两个特异性结合OX40的Fab片段以及两个特异性结合PD-L1的单结构域抗原结合位点(例如，VHH)，分别具有图1A－1D中例示的任一结构。所述两个特异性结合OX40的Fab片段特异性结合OX40分子上的相同表位或者不同表位；所述两个特异性结合PD-L1的单结构域抗原结合位点特异性结合PD-L1分子上的相同表位或者不同表位。
在一个实施方案中，本发明抗OX40/PD-L1双特异性抗体中的所述特异性结合OX40的Fab片段包含衍生自抗OX40抗体ADI-20112的SEQ ID NO:11/7的成对重链可变区序列/轻链可变区序列中所含的全部6个重链互补决定区(CDR)与轻链CDR，或者与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
在一个实施方案中，本发明抗OX40/PD-L1双特异性抗体中的所述特异性结合OX40的Fab片段包含衍生自抗OX40抗体ADI-20112的SEQ ID NO:11/7的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体中的所述特异性结合PD-L1的单结构域抗原结合位点包含SEQ ID NO:3所示的CDR1、SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
又在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体中的所述特异性结合PD-L1的单结构域抗原结合位点包含SEQ ID NO:1和/或SEQ ID NO:2所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
不特别地限制本发明抗OX40/PD-L1双特异性抗体中重链恒定区CH1结构域和Fc区(包含CH2结构域、CH3结构域和可选的CH4结构域)的类型，优选地是衍生自IgG1、IgG2或IgG4免疫球蛋白重链恒定区的相应结构域，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。更优选地，所述重链恒定区CH1结构域和Fc区衍生自人IgG1免疫球蛋白的重链恒定区的CH1结构域和Fc区， 或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体包含IgG4(例如，人IgG4)重链恒定区的CH1结构域和Fc区。在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体包含IgG1(例如，人IgG1)重链恒定区的CH1结构域和Fc区。又在一个实施方案中，本发明的抗OX40/PD-L1双特异性抗体包含IgG4(例如，人IgG4)重链恒定区的CH1结构域和IgG1(例如，人IgG1)重链恒定区的Fc区；或者包含IgG1(例如，人IgG1)重链恒定区的CH1结构域和IgG4(例如，人IgG4)重链恒定区的Fc区。
在一个实施方案中，本发明抗OX40/PD-L1双特异性抗体的第二多肽链和第四多肽链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，从而本发明抗OX40/PD-L1双特异性抗体的第二多肽链和第四多肽链在Fc区形成链间二硫键，由此，稳定第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明抗OX40/PD-L1双特异性抗体的第二多肽链和/或第四多肽链在Fc结构域中包含影响抗体效应子功能的氨基酸突变。在一个具体实施方案中，所述氨基酸置换是LALA突变。
在又一个实施方案中，本发明抗OX40/PD-L1双特异性抗体包含κ轻链恒定区和/或λ轻链恒定区，例如，人κ轻链恒定区和/或人λ轻链恒定区。在一个实施方案中，轻链恒定区包含在SEQ ID NO:8所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
在一个实施方案中，本发明抗OX40/PD-L1双特异性抗体的第二多肽链和第四多肽链在各自的Fc结构域中分别包含“结入扣”的稳定缔合。在一个实施方案中，在所述第二多肽链和第四多肽链之一条链中包含氨基酸置换T366W，并且在所述第二多肽链和第四多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(EU编号)。由此一条链中的凸起能够置于另一条链中的空穴中，促进第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明抗OX40/PD-L1双特异性抗体的免疫球蛋白CH1结构域和CL结构域中分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于CL结构域中的所述空穴或凸起中，从而本发明抗OX40/PD-L1双特异性抗体的第一多肽链和第二多肽链彼此也形成“结入扣”的稳定缔合。
在具体的实施方案中，本发明的抗OX40/PD-L1双特异性抗体由左右基本上对称的4条多肽链组成，其中所述抗体分子的左半部分的2条多肽链分别包含SEQ ID NO:6所示的第一多肽链和SEQ ID NO:10所示的第二多肽链；分别包含SEQ ID NO:14所示的第一多肽链和SEQ ID NO:10所示的第二多肽链；分别包含SEQ ID NO:15所示的第一多肽链和SEQ ID NO:16所示的第二多肽链；分别包含SEQ ID NO:15所示的第一多肽链和SEQ ID NO:17所示的第二多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列；相应地，其中所述抗体分子的 右半部分的2条多肽链分别包含SEQ ID NO:6所示的第三多肽链和SEQ ID NO:10所示的第四多肽链；分别包含SEQ ID NO:14所示的第三多肽链和SEQ ID NO:10所示的第四多肽链；分别包含SEQ ID NO:15所示的第三多肽链和SEQ ID NO:16所示的第四多肽链；分别包含SEQ ID NO:15所示的第三多肽链和SEQ ID NO:17所示的第四多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
本发明的抗OX40/PD-L1双特异性抗体能够同时与PD-L1和OX40蛋白结合，且维持了亲本抗体的亲和力常数，由此，能够阻断PD-1/PD-L1信号传导途径且活化T细胞和自然杀伤(NK)细胞中的OX40/OX40配体信号传导途径。本发明的抗OX40/PD-L1双特异性抗体能够用于与所述信号传导途径相关的疾病的治疗、预防或诊断。
ii)在一个实施方案中，本发明的抗体分子是抗VEGF/GITR双特异性抗体或多特异性抗体。
血管内皮生长因子(Vascular endothelial growth factor,VEGF)最初称为血管通透性因子(vascular permeability factor,VPF)(Senger,DR等人，Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid，Science，1983,219(4587):983–985)，是由刺激血管形成的细胞产生的信号蛋白。VEGF是生长因子的亚家族，其是一类参与血管生成的重要信号传导蛋白。在正常组织中血管内皮生长因子和血管内皮细胞生长抑制因子同时存在，且保持相对平衡，这种平衡使得人体血管可以正常地生成和分化。但是，在疾病例如肿瘤生长过程中，VEGF家族分子数量激增，与血管生成抑制因子之间的调节失衡，由此，极大地促进了血管内皮细胞的分裂增殖和迁移、提高了血管通透性、抑制肿瘤细胞凋亡，为肿瘤的生长和转移提供了良好的微环境(Lapeyre-Prost A等人，Immunomodulatory Activity of VEGF in Cancer，Int Rev Cell Mol Biol.2017；330:295-342)。VEGF家族包含六种密切相关的多肽，分别是高度保守的同源二聚体糖蛋白，有六个亚型：VEGF-A、-B、-C、-D、-E、和胎盘生长因子(placental growth factor(PLGF))，分子量从35至44kDa不等。VEGF-A(包括其剪接物如VEGF 165)的表达与一些实体瘤的微血管密度具有相关性，并且组织中VEGF-A的浓度与乳腺癌、肺癌、前列腺癌和结肠癌等实体瘤的预后有关。每个VEGF家族成员的生物学活性通过细胞表面VEGF受体(VEGFR)家族中的一种或多种介导，所述VEGFR家族包括VEGFR1(也称为Flt-1)、VEGFR2(也称为KDR、Flk-1)、VEGFR3(也称为Flt-4)等，其中VEGFR1、VEGFR2与血管的生成关系密切，VEGF-C/D/VEGFR3则与淋巴管生成密切相关。
临床研究显示利用抗VEGF单克隆抗体能够阻断VEGF与其受体的结合。基因泰克(Genentech)公司研发的贝伐单抗(Bevacizumab，商品名Avastin)是一种重组的人鼠嵌合抗VEGF抗体，可通过阻断VEGF-A与VEGFR的结合，使VEGFR无法活化，由此发挥抗血管生成的作用。贝伐单抗目前用于一线治疗转移性结直肠癌，将来有可能用于转移性肺癌、乳癌、胰脏癌、肾脏癌等疾病的治疗。贝伐单抗也是开发较为成功的抗体药物之一。
糖皮质激素诱导的肿瘤坏死因子受体(glucocorticoid induced tumor necrosis factor receptor，GITR，亦称作TNFRSF18、活化诱导型TNFR家族成员(AITR)、CD357及GITR-D)，是肿瘤坏死因子(tumor necrosis factor，TNF)受体超家族的第18个成员。最初在经地塞米松(dexamethasone)处理的鼠类T细胞系中鉴定出(Nocentini G等人，A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis，Proc Natl Acad Sci U S A.1997；94(12):6216-21)。TNF受体超家族的其他相关成员包括CD40、CD27、4-1BB和OX40。尽管GITR的表达在原初CD4+及CD8+细胞中较低，但其在调节性T细胞中组成型表达(Tone M等人，Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells，Proc Natl Acad Sci U S A.2003；100(25):15059-64)。但是，一旦在效应T细胞上诱导GITR表达，则促进效应T细胞活化、增殖及细胞因子产生。关于CD4+CD25+调节性T细胞(Treg)，Shimizu使用混合培养物阻抑分析报导GITR激活会阻抑Treg的功能(Shimizu J等人，Stimulation of CD25(+)CD4(+)regulatory T cells through GITR breaks immunological self-tolerance，Nature Immunology 2002；3:135-42)。在多种肿瘤模型中，抗GITR抗体DTA-1介导的GITR刺激促进抗肿瘤免疫(Cohen AD等人，Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation，PLoS One.2010；5(5):e10436；Coe D等人，Depletion of regulatory T cells by anti-GITR mAb as a novel mechanism for cancer immunotherapy，Cancer Immunol Immunother，2010；59(9):1367-77)。
GITR与GITR配体(GITRL)结合后活化，所述配体主要在APC上表达，GITR活化后能够增加对肿瘤和病毒感染的抗性，参与自身免疫过程/炎症性过程，并调节白细胞外渗(Cohen AD等人，同上；和 Cuzzocrea S等人，Genetic and pharmacological inhibition of GITR-GITRL interaction reduces chronic lung injury induced by bleomycin instillation， FASEB J.2007，21(1):117-129)。
以下文献中描述了抗GITR抗体：美国专利号7,025,962、欧洲专利号1947183B1、美国专利号7,812,135、美国专利号8,388,967、美国专利号8,591,886、欧洲专利号EP 1866339、PCT公开号WO 2011/028683、美国专利号8,709,424、PCT公开号WO 2013/039954、国际公开号WO 2013/039954、美国公开号US 2014/0072566、国际公开号WO 2015/026684、PCT公开号WO 2005/007190、PCT公开号WO 2007/133822、PCT公开号WO 2005/055808、PCT公开号WO 99/40196、PCT公开号WO 2001/03720、PCT公开号WO 99/20758、美国专利号6,689,607、PCT公开号WO 2006/083289、PCT公开号WO 2005/115451、美国专利号7,618,632、PCT公开号WO 2011/051726、国际公开号WO 2004060319和国际公开号WO2014012479。
本发明的抗VEGF/GITR双特异性抗体或多特异性抗体至少同时靶向VEGF和GITR，其Fab片段和单结构域抗原结合位点分别结合VEGF或GITR分子，能够阻断VEGF家族信号传导途径以及活化效应T细胞和阻抑Treg的功能。
在一个实施方案中，本发明的抗体分子包含特异性结合GITR的单结构域抗原结合位点和特异性结合VEGF的Fab片段。在一个实施方案中，本发明的抗体分子包含特异性结合VEGF的单结构域抗原结合位点和特异性结合GITR的Fab片段。
对于所述特异性结合GITR或VEGF的Fab片段，其包含衍生自任何现有技术中报导的抗GITR抗体(例如，上文中例举的抗GITR抗体)和将来研发出的抗GITR抗体VH/VL对的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；或者包含衍生自任何现有技术中报导的抗VEGF抗体(例如，上文中例举的抗VEGF抗体)和将来研发出的抗VEGF抗体VH/VL对的6个CDR或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。在一个实施方案中，抗VEGF抗体是Avastin，其具有SEQ ID NO:19所示的重链氨基酸序列和SEQ ID NO:18所示的轻链氨基酸序列。
对于所述特异性结合GITR或VEGF的单结构域抗原结合位点，其包含特异性结合GITR或VEGF的重链可变结构域(VH)、轻链可变结构域(VL)、来自骆驼科血清的天然不含轻链的仅由两条重链组成的骆驼抗体中的重链可变结构域、来自鲨鱼科动物的IgNAR的VH样单结构域、骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域。
在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体包含两个特异性结合VEGF的Fab片段以及两个特异性结合GITR的单结构域抗原结合位点(例如，VHH)，分别具有图1A、图1B、图1D、图11A和图11B中例示的任一结构。所述两个特异性结合VEGF的Fab片段特异性结合VEGF分子上的相同表位或者不同表位；所述两个特异性结合GITR的单结构域抗原结合位点特异性结合GITR分子上的相同表位或者不同表位。
在一个实施方案中，本发明抗VEGF/GITR双特异性抗体中的所述特异性结合VEGF的Fab片段包含衍生自抗VEGF抗体Avastin的SEQ ID NO:22/20的成对重链可变区序列/轻链可变区序列中所含的全部6个重链互补决定区(CDR)与轻链CDR，或者与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
在一个实施方案中，本发明抗VEGF/GITR双特异性抗体中的所述特异性结合VEGF的Fab片段包含衍生自抗VEGF抗体Avastin的SEQ ID NO:22/20的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。
在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体中的所述特异性结合GITR的单结构域抗原结合位点包含GFAFGSS(SEQ ID NO:25)所示的CDR1、SGGGFGD(SEQ ID NO:26)所示的CDR2和ATDWRKP(SEQ ID NO:27)所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。
又在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体中的所述特异性结合GITR的单结构域抗原结合位点包含SEQ ID NO:24所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
不特别地限制本发明抗VEGF/GITR双特异性抗体中重链恒定区CH1结构域和Fc区(包含CH2结构域、CH3结构域和可选的CH4结构域)的类型，优选地是衍生自IgG1、IgG2或IgG4免疫球蛋白重链恒定区的相应结构域，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。更优选地，所述重链恒定区CH1结构域和Fc区衍生自人IgG1免疫球蛋白的重链恒定区的CH1结构域和Fc区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体包含IgG4(例如，人IgG4)重链恒定区的CH1结构域和Fc区。在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体包含IgG1(例如，人IgG1)重链恒定区的CH1结构域和Fc区。又在一个实施方案中，本发明的抗VEGF/GITR双特异性抗体包含IgG4(例如，人IgG4)重链恒定区的CH1结构域和IgG1(例如，人IgG1)重链恒定区的Fc区；或者包含IgG1(例如，人IgG1)重链恒定区的CH1结构域和IgG4(例如，人IgG4)重链恒定区的Fc区。
在一个实施方案中，本发明抗VEGF/GITR双特异性抗体的第二多肽链和第四多肽链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，由此，本发明抗VEGF/GITR双特异性抗体的第二多肽链和第四多肽链在Fc区形成链间二硫键，由此，稳定第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明抗VEGF/GITR双特异性抗体的第二多肽链和/或第四多肽链在Fc结构域中包含影响抗体效应子功能的氨基酸突变。在一个具体实施方案中，所述氨基酸置换是LALA突变。
在又一个实施方案中，本发明抗VEGF/GITR双特异性抗体包含κ轻链恒定区和/或λ轻链恒定区，例如，人κ轻链恒定区和/或人λ轻链恒定区。在一个实施方案中，轻链恒定区包含在SEQ ID NO:8所示的氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。
在一个实施方案中，本发明抗VEGF/GITR双特异性抗体的第二多肽链和第四多肽链在各自的Fc结构域中分别包含“结入扣”的稳定缔合。在一个实施方案中，在所述第二多肽链和第四多肽链之一条链中包含氨基酸置换T366W，并且在所述第二多肽链和第四多肽链之另一条链中包含氨基酸置换T366S、L368A和Y407V(EU编号)。由此一条链中的凸起能够置于另一条链中的空穴中，促进第二多肽链和第四多肽链的正确配对。
在一个实施方案中，本发明抗VEGF/GITR双特异性抗体的免疫球蛋白CH1结构域和CL结构域中分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于 CL结构域中的所述空穴或凸起中，从而本发明抗VEGF/GITR双特异性抗体的第一多肽链和第二多肽链彼此也形成“结入扣”的稳定缔合。
在具体的实施方案中，本发明的抗VEGF/GITR双特异性抗体由左右基本上对称的4条多肽链组成，其中所述抗体分子的左半部分的2条多肽链分别包含SEQ ID NO:18所示的第一多肽链和SEQ ID NO:21所示的第二多肽链；分别包含SEQ ID NO:18所示的第一多肽链和SEQ ID NO:28所示的第二多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列；相应地，其中所述抗体分子的右半部分的2条多肽链分别包含SEQ ID NO:18所示的第三多肽链和SEQ ID NO:21所示的第四多肽链；分别包含SEQ ID NO:18所示的第三多肽链和SEQ ID NO:28所示的第四多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
本发明的抗VEGF/GITR双特异性抗体能够同时与GITR和VEGF蛋白结合，且维持了亲本抗体的亲和力常数，由此，能够阻断VEGF家族信号传导途径以及活化效应T细胞和自然杀伤(NK)细胞中的GITR/GITR配体信号传导途径。本发明的抗VEGF/GITR双特异性抗体能够用于与所述信号传导途径相关的疾病的治疗、预防或诊断。
III.本发明的抗体分子变体
在某些实施方案中，构思了本文例示的双特异性抗体的氨基酸序列变体。例如，可能想要改善双特异性抗体的结合亲和力和/或其他生物学特性。可以通过向编码双特异性抗体的核苷酸序列引入适宜修饰或通过肽合成制备双特异性抗体的氨基酸序列变体。此类修饰包括例如从抗体的氨基酸序列内部缺失残基和/或将残基插入所述氨基酸序列中和/或置换所述氨基酸序列中的残基。可以产生缺失、插入和置换的任意组合以获得最终构建体，只要所述最终构建体拥有想要的特征，例如抗原结合作用。
表1中在“保守性置换”标题下显示保守性置换。表1中在“示例性置换”标题下显示并且参考氨基酸侧链类别如下文进一步描述更明显的变化。可以将氨基酸置换引入目的抗体中并且对产物筛选所需的活性，例如，保留/改善的抗原结合作用或降低的免疫原性。
表1
Figure PCTCN2019079671-appb-000002
Figure PCTCN2019079671-appb-000003
氨基酸可以根据常见的侧链特性分组：
(1)疏水性：正亮氨酸、Met、Ala、Val、Leu；Ile；
(2)中性亲水：Cys、Ser、Thr、Asn；Gln；
(3)酸性：Asp、Glu；
(4)碱性：His、Lys、Arg；
(5)影响链方向的残基：Gly、Pro；
(6)芳族：Trp、Tyr、Phe。
非保守性置换将使这些分类之一的成员交换为另一个分类的成员。
IV.免疫缀合物
本发明的抗体分子能够重组融合于或化学缀合(包括共价和非共价缀合)至异源蛋白或多肽以产生融合蛋白。蛋白质、多肽或肽与抗体融合或缀合的方法是本领域已知的。参见，例如，美国专利号5,336,603、5,622,929和EP 367,166。
另外，本发明的抗体分子可以与标记序列(如肽)融合以促进纯化。在优选的实施方案中，标记氨基酸序列是六组氨酸肽，如pQE载体(QIAGEN,Inc.,9259 Eton Avenue,Chatsworth,CA,91311)等中提供的标签，它们中的许多是可商业获得的。如Gentz等人,1989,Proc.Natl.Acad.Sci.USA 86:821-824中所述，例如，六组氨酸提供融合蛋白的便利纯化。用于纯化的其他肽标签包括但不限于血凝素(“HA”)标签，其对应于源自流感血凝素蛋白的表位(Wilson等人,1984,Cell 37:767)和“flag”标签。
在其他实施方案中，本发明的抗体分子与诊断剂或可检测剂缀合。这类抗体可以作为临床检验方法的部分(如确定特定疗法的效力)，用于监测或预测疾病或病症的发作、形成、进展和/或严重性。可以通过将抗体与可检测物质偶联实现这类诊断和检测，所述可检测物质包括但不限于多种酶，如但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶 或乙酰胆碱酯酶；辅基，如但不限于链霉亲和素/生物素和抗生物素蛋白/生物素；荧光物质，如但不限于伞形酮、荧光素、异硫氰酸荧光素、罗丹明、二氯三嗪胺荧光素、丹磺酰氯或藻红蛋白；发光物质，如但不限于鲁米诺；生物发光物质，如但不限于萤光素酶、萤光素和水母发光蛋白；放射性物质，如但不限于碘( 131I、 125I、 123I和 121I)、碳( 14C)、硫( 35S)、氚( 3H)、铟( 115In、 113In、 112In和 111In)、锝( 99Tc)、铊( 201Ti)、镓( 68Ga、 67Ga)、钯( 103Pd)、钼( 99Mo)、氙( 133Xe)、氟( 18F)、 153Sm、 177Lu、 159Gd、 149Pm、 140La、 175Yb、 166Ho、 90Y、47Sc、 186Re、 188Re、 142Pr、 105Rh、 97Ru、 68Ge、 57Co、 65Zn、 85Sr、 32P、 153Gd、 169Yb、 51Cr、 54Mn、 75Se、 113Sn和 117Tin；和用于各种正电子发射成像术中的正电子发射金属和非放射性顺磁金属离子。
本发明还包括与治疗性部分缀合的抗体分子的用途。抗体分子可缀合到治疗性部分，如细胞毒素(例如细胞生长抑制剂或细胞杀伤剂)，治疗剂或放射性金属离子，例如α发射体。术语“细胞毒素”或“细胞毒性剂”包括有害于细胞的任何物质。
另外，本发明的抗体分子可以与调节给定生物学反应的治疗性部分或药物部分缀合。治疗性部分或药物部分不得解释为限于经典的化学治疗药。例如，药物部分可以是拥有所需生物学活性的蛋白质、肽或多肽。这类蛋白质可以例如包括毒素如相思豆毒蛋白、蓖麻毒蛋白A、假单胞菌外毒素、霍乱毒素、或白喉毒素；蛋白质如肿瘤坏死因子、α-干扰素、β-干扰素、神经生长因子、血小板衍生生长因子、组织纤维蛋白溶酶原激活物、凋亡剂、抗血管生成剂或生物学反应调节物，例如淋巴因子。
另外，本发明的抗体分子可以缀合至治疗性部分如放射性金属离子，如α-发射体如 213Bi或可用于使放射金属离子(包括但不限于 131In、 131LU、 131Y、 131Ho、 131Sm)缀合至多肽的大环螯合剂。在某些实施方案中，大环螯合剂是1,4,7,10-四氮杂环十二烷-N,N’,N”,N”’-四乙酸(DOTA)，其可通过接头分子附着到抗体上。这类接头分子是本领域公知的并且在Denardo等人,1998,Clin Cancer Res.4(10):2483-90中描述，所述文献每篇通过引用的方式完整并入。
用于治疗性部分与抗体缀合的技术是熟知的，参见，例如Arnon等人,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”，引自Monoclonal Antibodies And Cancer Therapy,Reisfeld等人(编著)，第243-256页(Alan R.Liss,Inc.1985)。
抗体也可以连接至固相支持物，所述支持物特别可用于免疫测定法或靶抗原的纯化。此类固相支持物包括但不限于玻璃、纤维素、聚丙烯酰胺、尼龙、聚苯乙烯、聚氯乙烯或聚丙烯。
V.本发明的抗体分子的生产和纯化
本发明的抗体分子可以例如通过固态肽合成(例如Merrifield固相合成)或重组生产获得。为了重组生产，将编码所述抗体分子的任意一条多肽链和/或多条多肽链的多核苷酸分离并插入一个或多个载体中以便进一步在宿主细胞中克隆和/或表达。使用常规方法，可以轻易地分离所述多核苷酸并将其测序。在一个实施方案中，提供了包含本发明的一种或多种多核苷酸的载体，优选地表达载体。
可以使用本领域技术人员熟知的方法来构建表达载体。表达载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。
一旦已经制备了用于表达的包含本发明的一种或多种多核苷酸的表达载体，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质体的转染或其他常规技术。
在一个实施方案中，提供了包含一种或多种本发明多核苷酸的宿主细胞。在一些实施方案中，提供了包含本发明表达载体的宿主细胞。如本文所用，术语“宿主细胞”指可以工程化以产生本发明的抗体分子的任何种类的细胞系统。适于复制和支持本发明的抗体分子表达的宿主细胞是本领域熟知的。根据需要，这类细胞可以用特定表达载体转染或转导，并且可以培育大量含有载体的细胞用于接种大规模发酵器以获得足够量的本发明抗体分子用于临床应用。合适的宿主细胞包括原核微生物，如大肠杆菌，真核微生物如丝状真菌或酵母，或各种真核细胞，如中国仓鼠卵巢细胞(CHO)、昆虫细胞等。可以使用适于悬浮培养的哺乳动物细胞系。有用的哺乳动物宿主细胞系的例子包括SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK 293或293F细胞)、幼仓鼠肾细胞(BHK)、猴肾细胞(CV1)、非洲绿猴肾细胞(VERO-76)、人宫颈癌细胞(HELA)、犬肾细胞(MDCK)、布法罗大鼠肝脏细胞(BRL 3A)、人肺细胞(W138)、人肝脏细胞(Hep G2)、CHO细胞、NSO细胞、骨髓瘤细胞系如YO、NS0、P3X63和Sp2/0等。适于产生蛋白质的哺乳动物宿主细胞系的综述参见例如Yazaki和Wu，Methods in Molecular Biology，第248卷(B.K.C.Lo编著，Humana Press，Totowa，NJ)，第255-268页(2003)。在一个优选的实施方案中，所述宿主细胞是CHO、HEK293或NSO细胞。
本领域已知在这些宿主细胞系统中表达外源基因的标准技术。在一个实施方案中，提供了产生本发明的抗体分子的方法，其中所述方法包括在适于表达所述抗体分子的条件下培养如本文中提供的宿主细胞，所述宿主细胞包含编码所述抗体分子的多核苷酸，并且从宿主细胞(或宿主细胞培养基)回收所述抗体分子。
如本文所述制备的抗体分子可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于如净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。
可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。可以通过本领域已知的多种测定法，鉴定、筛选或表征本文提供的抗体分子的物理/化学特性和/或生物学活性。
VI.药物组合物和试剂盒
在另一个方面，本发明提供了组合物，例如，药物组合物，所述组合物包含与可药用载体配制在一起的本文所述的抗体分子。如本文所用，“可药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。本发明的药物组合物适于静脉内、肌内、皮下、肠胃外、直肠、脊髓或表皮施用(例如，通过注射或输注)。
本文中还公开了本文所述的抗体分子与一种以上治疗剂组合后获得的组合物，所述治疗剂选自以下类别(i)-(iii)中的一个、两个或全部类别：(i)增强抗原呈递(例如，肿瘤抗原呈递)的药物；(ii)增强效应细胞反应(例如，B细胞和/或T细胞活化和/或动员)的药物；或(iii)减少免疫抑制的药物。
本发明的组合物可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，可注射用溶液剂和可输注溶液剂)、分散体剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。常见的优选组合物处于可注射用溶液剂或可输注溶液剂形式。优选的施用模式是肠胃外(例如，静脉内、皮下、腹腔(i.p.)、肌内)注射。在一个优选实施方案中，通过静脉内输注或注射施用抗体分子。在另一个优选实施方案中，通过肌内、腹腔或皮下注射施用抗体分子。
如本文所用的短语“肠胃外施用“和“肠胃外方式施用”意指除了肠施用和局部施用之外的施用模式，通常通过注射施用，并且包括但不限于静脉内、肌内、动脉内、皮内、腹腔、经气管、皮下注射和输注。
治疗性组合物一般应当是无菌的并且在制造和储存条件下稳定。可以将组合物配制为溶液、微乳液、分散体、脂质体或冻干形式。可以通过将活性化合物(即抗体分子)以要求的量加入适宜的溶剂中，随后过滤消毒，制备无菌可注射溶液剂。通常，通过将所述活性化合物并入无菌溶媒中来制备分散体，所述无菌溶媒含有基础分散介质和其他成分。可以使用包衣剂如卵磷脂等。在分散体的情况下，可以通过使用表面活性剂来维持溶液剂的适宜流动性。可以通过在组合物中包含延迟吸收的物质例如单硬脂酸盐和明胶而引起可注射组合物的延长吸收。
在某些实施方案中，可以口服施用本发明的抗体分子，例如随惰性稀释剂或可食用载体一起经口施用。本发明的抗体分子也可以封闭在硬壳或软壳明胶胶囊中、压缩成片剂或直接掺入受试者的膳食中。对于口服治疗施用，所述化合物可以随赋形剂一起掺入并且以可摄取的片剂、颊用片剂、锭剂(troche)、胶囊剂、酏剂、混悬剂、糖浆剂、糯米纸囊剂(wafer)等形式使用。为了通过非肠胃外施用方法施用本发明的抗体分子，可能需要将所述抗体分子与防止其失活的材料包衣或随这种材料共施用。还可以用本领域已知的医疗装置施用治疗组合物。
本发明的药物组合物可以包含“治疗有效量”或“预防有效量”的本发明所述抗体分子。“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。可以根据多种因素如疾病状态、个体的年龄、性别和重量等变动治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的受试者，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约60％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的抗体分子抑制可度量参数(例如，肿瘤体积)的能力。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用，故预防有 效量小于治疗有效量。
包含本文所述抗体分子的试剂盒也处于本发明的范围内。试剂盒可以包含一个或多个其他要素，例如包括：使用说明书；其他试剂，例如标记物或用于偶联的试剂；可药用载体；和用于施用至受试者的装置或其他材料。
VII.抗体分子的用途
本文公开的抗体分子具有体外和体内诊断用途以及治疗性和预防性用途。例如，可以将这些分子施用至体外或离体的培养细胞或施用至受试者，例如，人类受试者，以治疗、预防和/或诊断多种抗原相关的疾病，例如癌症、自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)。
在一个方面，本发明提供了体外或体内检测生物样品，例如血清、精液或尿或组织活检样品(例如，来自过度增生性或癌性病灶)中存在相关抗原的诊断方法。该诊断方法包括：(i)在允许相互作用发生的条件下使样品(和任选地，对照样品)与如本文所述的抗体分子接触或向受试者施用所述抗体分子和(ii)检测所述抗体分子和样品(和任选地，对照样品)之间复合物的形成。复合物的形成表示存在相关抗原，并且可以显示本文所述治疗和/或预防的适用性或需求。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测相关抗原。可以使用的检测方法包括免疫组织化学、免疫细胞化学、FACS、ELISA测定、PCR技术(例如，RT-PCR)或体内成像技术。一般地，体内和体外检测方法中所用的抗体分子直接或间接地用可检测物质标记以促进检测结合的或未结合的结合物。合适的可检测物质包括多种生物学活性酶、辅基、荧光物质、发光物质、顺磁(例如，核磁共振活性)物质和放射性物质。
在一些实施方案中，体内确定相关抗原的水平和/或分布，例如，以非侵入方式确定(例如，通过使用合适的成像技术(例如，正电子发射断层摄影术(PET)扫描)检测可检测物标记的本发明抗体分子。在一个实施方案中，例如，通过检测用PET试剂(例如， 18F-氟脱氧葡萄糖(FDG))以可检测方式标记的本发明抗体分子，体内测定相关抗原的水平和/或分布。
在一个实施方案中，本发明提供了包含本文所述抗体分子和使用说明书的诊断试剂盒。
在另一个方面，本发明涉及使用本发明的抗体分子体内用来治疗或预防需要在受试者中调节免疫应答的疾病，从而抑制或减少相关疾病如癌性肿瘤、自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)的出现或复发。可以单独使用本发明的抗体分子。备选地，抗体分子可以与其他癌症治疗剂/预防剂组合施用。当本发明的抗体分子与一种或多种其他药物组合施用时，这种组合可以按任何顺序施用或者同时施用。
因此，在一个实施方案中，本发明提供一种调节受试者中免疫应答的方法，所述方法包括向受试者施用治疗有效量的本文所述的抗体分子。在另一个实施方案中，本发明 提供一种防止受试者中疾病出现或者复发的方法，所述方法包括向受试者施用预防有效量的本文所述的抗体分子。
在一些实施方案中，用抗体分子治疗和/或预防的癌包括但不限于实体瘤、血液学癌(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓瘤)及转移性病灶。在一个实施方案中，癌是实体瘤。实体瘤的例子包括恶性肿瘤，例如，多个器官系统的肉瘤和癌，如侵袭肺、乳房、卵巢、淋巴样、胃肠道的(例如，结肠)、肛门、生殖器和生殖泌尿道(例如，肾、膀胱上皮、膀胱细胞、前列腺)、咽、CNS(例如，脑、神经的或神经胶质细胞)、头和颈、皮肤(例如，黑素瘤)、鼻咽(例如，分化或未分化的转移性或局部复发性鼻咽癌)和胰的那些癌、以及腺癌，包括恶性肿瘤，如结肠癌、直肠癌、肾细胞癌、肝癌、非小细胞肺癌、小肠癌和食道癌。癌症可以处于早期、中期或晚期或是转移性癌。
在一些实施方案中，癌选自黑素瘤、乳腺癌、结肠癌、食管癌、胃肠道间质肿瘤(GIST)、肾癌(例如，肾细胞癌)、肝癌、非小细胞肺癌(NSCLC)、卵巢癌、胰腺癌、前列腺癌、头颈部肿瘤、胃癌、血液学恶性病(例如，淋巴瘤)。
在一些实施方案中，用抗体分子治疗和/或预防的感染性疾病包括目前不存在有效疫苗的病原体或常规疫苗对其未及完全有效的病原体。这些包括但不限于HIV、(甲型、乙型和丙型)肝炎、流感、疱疹、贾弟鞭毛虫(Giardia)、疟疾、利什曼原虫(Leishmania)、金黄色葡萄球菌(Staphylococcus aureus)、铜绿假单胞菌(Pseudomonas aeruginosa)。本发明例示的抗体分子对PD-L1阻断作用特别可用来对抗随感染过程推移出现变异抗原的病原体(如HIV)所建立的感染。这些变异抗原在施用抗人PD-L1抗体时能够被视为外来抗原，由此，本发明例示的抗体分子能够通过PD-L1激发不受负向信号抑制的强烈T细胞反应。
在一些实施方案中，用本发明的抗体分子治疗和/或预防炎性和自身免疫性疾病及移植物抗宿主病(GvHD)，来下调免疫系统。可以通过施用本发明抗体分子治疗和/或预防的自身免疫疾病的例子包括但不限于斑秃、强直性脊柱炎、自身免疫性肝炎节段性回肠炎、红斑狼疮、溃疡性结肠炎、葡萄膜炎等。可以通过施用本发明抗体分子治疗和/或预防的炎性疾病的例子包括但不限于哮喘、脑炎、炎性肠病、过敏性疾病、败血性休克、肺纤维化、关节炎和因慢性病毒性或细菌性感染产生的慢性炎症。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1.抗OX40/PD-L1双特异性抗体的构建、表达、纯化及性质鉴定
实施例1.1.抗OX40/PD-L1双特异性抗体的构建
在本实施例中，构建了4种结构的抗OX40/PD-L1双特异性抗体，分别命名为(1)双特异性抗体Bi-110-112HC，其结构示意图如图1A所示；(2)双特异性抗体Bi-113-112HC，其结构示意图如图1B所示；(3)双特异性抗体Bi-119-112LC，其结构示意图如图1C所示；和(4)双特异性抗体Bi-122-112LC，其结构示意图如图1D所示。下面对这四种抗 OX40/PD-L1双特异性抗体分别进行描述。
(1)从图1A的结构示意图可见，双特异性抗体Bi-110-112HC由左右对称的4条多肽链组成，其中左半部分的2条多肽链(即，肽链#1和肽链#2)从N端至C端分别具有SEQ ID NO:6和SEQ ID NO:10所示的氨基酸序列。具体而言，在SEQ ID NO:6所示的肽链#1中从N端至C端包含衍生自抗OX40抗体ADI-20112的SEQ ID NO:7所示的VL氨基酸序列、在所述VL氨基酸序列C端的SEQ ID NO:8所示的人κ轻链恒定区(CL)氨基酸序列、在所述人κ轻链恒定区(CL)氨基酸序列C端的SEQ ID NO:9所示的连接肽氨基酸序列、以及在所述连接肽氨基酸序列C端的SEQ ID NO:2所示的抗PD-L1 VHH氨基酸序列。在SEQ ID NO:10所示的肽链#2中包含衍生自抗OX40单克隆抗体ADI-20112的SEQ ID NO:11所示的VH氨基酸序列、在所述VH氨基酸序列C端的衍生自人IgG1的SEQ ID NO:12所示的CH1氨基酸序列、以及在所述CH1氨基酸序列C端的衍生自人IgG1的SEQ ID NO:13所示的Fc区氨基酸序列。
(2)从图1B的结构示意图可见，双特异性抗体Bi-113-112HC由左右对称的4条多肽链组成，其中左半部分的2条多肽链(即，肽链#1和肽链#2)从N端至C端分别具有SEQ ID NO:14和SEQ ID NO:10所示的氨基酸序列。具体而言，在SEQ ID NO:14所示的肽链#1中从N端至C端包含SEQ ID NO:2所示的抗PD-L1VHH氨基酸序列、SEQ ID NO:9所示的连接肽氨基酸序列、SEQ ID NO:7所示的衍生自抗OX40抗体ADI-20112的VL氨基酸序列、以及SEQ ID NO:8所示的人κ轻链恒定区(CL)氨基酸序列。肽链#2具有SEQ ID NO:10所示的氨基酸序列。
(3)从图1C的结构示意图可见，双特异性抗体Bi-119-112LC由左右对称的4条多肽链组成，其中左半部分的2条多肽链(即，肽链#1和肽链#2)从N端至C端分别具有SEQ ID NO:15和SEQ ID NO:16所示的氨基酸序列。具体而言，在SEQ ID NO:15所示的肽链#1中从N端至C端包含SEQ ID NO:7所示的衍生自抗OX40抗体ADI-20112的VL氨基酸序列和SEQ ID NO:8所示的人κ轻链恒定区(CL)氨基酸序列。在SEQ ID NO:16所示的肽链#2中从N端至C端包含SEQ ID NO:11所示的衍生自抗OX40单克隆抗体ADI-20112的VH氨基酸序列、衍生自人IgG1的CH1氨基酸序列、SEQ ID NO:2所示的抗PD-L1VHH氨基酸序列和SEQ ID NO:13所示的衍生自人IgG1Fc区的氨基酸序列。
(4)从图1D的结构示意图可见，双特异性抗体Bi-122-112LC由左右对称的4条多肽链组成，其中左半部分的2条多肽链(即，肽链#1和肽链#2)从N端至C端分别具有SEQ ID NO:15和SEQ ID NO:17所示的氨基酸序列，其中，所述肽链#2从N端至C端包含SEQ ID NO:2所示的抗PD-L1VHH氨基酸序列、SEQ ID NO:9所示的连接肽氨基酸序列、SEQ ID NO:11所示的衍生自抗OX40单克隆抗体ADI-20112的VH氨基酸序列、SEQ ID NO:12所示的衍生自人IgG1的CH1氨基酸序列、和SEQ ID NO:13所示的衍生自人IgG1 Fc区的氨基酸序列。
实施例1.2.抗OX40/PD-L1双特异性抗体的表达、纯化和分析
在本实施例中，将编码实施例1.1中构建的抗OX40/PD-L1双特异性抗体的肽链#1、 肽链#2的核苷酸序列分别通过多克隆位点连接入市售的真核表达载体pTT5，在真核细胞中进行表达和纯化，获得了抗OX40/PD-L1双特异性抗体Bi-110-112HC、Bi-113-112HC、Bi-119-112LC和Bi-122-112LC。具体操作如下。
委托苏州金唯智生物科技有限公司(Genewiz)合成了双特异性抗体Bi-110-112HC、Bi-113-112HC、Bi-119-112LC和Bi-122-112LC的上述各肽链的编码核苷酸序列。使用合适的限制性内切酶和连接酶将所合成的编码肽链的核苷酸序列分别连接入载体pTT5中，获得了分别含有所述编码肽链的核苷酸序列的重组载体。
所述重组载体经测序验证正确后用于随后的表达。
将HEK293细胞(购自Invitrogen公司)传代培养于Expi293细胞培养液(购自Invitrogen公司)中。转染前一天离心细胞培养物，获得细胞沉淀，用新鲜的Expi293细胞培养液悬浮细胞，将细胞密度调整为1×10 6个细胞/ml。继续培养HEK293细胞，使得转染当天的培养物中细胞密度约为2×10 6个细胞/ml。取HEK293细胞悬浮液终体积1/10的F17培养基(购自Gibco公司，产品目录号A13835-01)作为转染缓冲液。向每毫升转染缓冲液中加入200μg的1:1摩尔比率的上述制备的分别包含编码肽链#1或肽链#2的核苷酸序列的重组质粒，混匀，再加入聚乙烯亚胺(polyethylenimine(PEI))(Polysciences，目录号：23966)30μg，混匀，室温温育10分钟后，将PEI/DNA混合物轻柔倒入HEK293细胞悬浮液中。轻轻混匀，置于8％CO 2、36.5℃过夜培养。
过夜培养后，向培养瓶中补加转染后培养物体积1/50的浓度为200g/L的FEED(Sigma，目录号：H6784-100G)和转染后培养物体积1/50的浓度为200g/L的葡萄糖溶液，轻轻混匀，置于8％CO 2、36.5℃继续培养。20小时后，加入VPA(Gibco，目录号：11140-050)至终浓度为2mM/L。连续培养至第7天或者细胞活力≤60％时，收集培养物，以7500转/分钟离心30分钟，取细胞上清，使用SARTOPORE(Sartorius，目录号：5441307H4)过滤后，在AKTApure系统(GE Healthcare)上通过亲和层析进行纯化。
具体的亲和层析纯化操作步骤为：选用MabSelect SuRe(GE Healthcare,目录号：17-5438-03)亲和层析柱，并置于AKTApure系统内。用0.1M NaOH对装备有MabSelect SuRe亲和层析柱的AKTApure系统过夜除内毒，然后用5倍柱体积的结合缓冲液(Tris 20mM，NaCl 150mM，pH 7.2)清洗系统以及平衡柱子。将上述过滤后的细胞上清通过柱子。用5至10倍柱体积的结合缓冲液再平衡，使用AKTApure系统配备的紫外检测装置监测至紫外走平。然后，用洗脱缓冲液(柠檬酸+柠檬酸钠100mM，pH 3.5)洗脱抗体，根据紫外吸收值来收集样品。每1ml的收集液加80μl的中和缓冲液(Tris-HCl 2M)中和备用。
利用大小排阻层析(size exclusion chromatography；SEC)检测收集的各级分管中样品的纯度。SEC结果分别如图2A、图2B、图2C和图2D所示，双特异性抗体Bi-110-112HC纯度为71.40％，Bi-113-112HC纯度为84.54％，Bi-119-112LC纯度为99.43％，Bi-122-112LC纯度为94.79％。
将纯化后的双特异性抗体溶液使用15ml超滤离心管，4500转/分钟离心30分钟。 使用PBS将蛋白稀释后继续离心，4500转/分钟离心30分钟，重复该操作2次，以更换缓冲液。将更换缓冲液后的抗体合并，测抗体浓度。
在后续实验中选取单体主峰纯度为99.43％的双特异性抗体Bi-119-112LC进行进一步的研究。
实施例1.3.测定抗OX40/PD-L1双特异性抗体的解离常数
使用Octet系统(ForteBio公司生产)通过动力学结合测定法确定本发明的上述示例性抗OX40/PD-L1双特异性抗体Bi-119-112LC结合OX40和PD-L1的平衡解离常数(K D)。按照文献中报导的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013，5(2):p.270-278)进行ForteBio亲和力测定。简言之，在实验开始前半个小时，将AHC传感器(Pall，目录号：1506091)浸泡于SD缓冲液(PBS 1×，BSA 0.1％，吐温20 0.05％)中于室温平衡。向96孔黑色聚苯乙烯半量微孔板(Greiner)的孔中分别加入100μl的SD缓冲液作为空白对照(用于扣除背景)、100μl 100nM纯化的双特异性抗体Bi-119-112LC和作为对照的抗PD-L1人源化Nb-Fc抗体(PCT/CN2017/095884)、抗OX40抗体ADI-20112(中国发明专利申请号201710185400.8)、100μl稀释于SD缓冲液中作为抗原的人PD-L1-his(100nM)和人OX40-his(100nM)(Acrobiosystems)的溶液。将抗人IgG Fc生物传感器AHC浸没于分别含所述抗体溶液的孔中，在室温浸没600秒上样。随后将传感器在SD缓冲液中洗涤至达到基线，然后浸没于含100μl抗原溶液的孔中，监测抗体与抗原的结合。随后将传感器转移至含有100μl SD缓冲液的孔，监测抗体解离。转速为400转/分钟，温度为30℃。通过Octet分析软件(ForteBio)拟合经背景校正的结合曲线和解离曲线，产生结合(k on)和解离(k dis)速率常数，它们随后用来计算平衡解离常数(K D)。表1和表2中显示了双特异性抗体Bi-119-112LC与抗原OX40或PD-L1的k on、k dis和K D数据。
表1.通过ForteBio动力学结合测定法确定的抗OX40/PD-L1双特异性抗体对OX40的亲和力
Figure PCTCN2019079671-appb-000004
表2.通过ForteBio动力学结合测定法确定的抗OX40/PD-L1双特异性抗体对PD-L1的亲和力
Figure PCTCN2019079671-appb-000005
Figure PCTCN2019079671-appb-000006
通过以上数据可见，本发明的双特异性抗体Bi-119-112LC能够同时与溶液中的PD-L1和OX40蛋白结合，且维持了亲本抗体ADI-20112和人源化Nb-Fc与各相应抗原的亲和力常数。
实施例1.4.本发明的抗OX40/PD-L1双特异性抗体与过量表达OX40或PD-L1的CHO细胞的结合分析
通过FACS测量了本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC与过量表达OX40或PD-L1的CHO细胞的结合。
简而言之，使用ExpiCHO TM Expression System Kit(Invitrogen，目录号：A29133)，根据制造商的说明书实施如下操作：将携带克隆至多克隆位点MCS的人PD-L1 cDNA(Sino Biological Inc.)的pCHO1.0载体(Invitrogen)转染至中国仓鼠卵巢癌细胞(CHO)(Invitrogen)，产生过量表达人PD-L1的CHO细胞(CHO-PD-L1细胞)。将CHO-PD-L1细胞计数，用细胞培养基稀释至1×10 6个细胞/ml,向U型底96孔板中以100μl/孔加入。在离心机上以400g离心5分钟，去除细胞培养基。分别将100μl系列稀释的本发明的双特异性抗体Bi-119-112LC和作为对照的人源化Nb-Fc加入U型板并重悬细胞，冰上静置30分钟。400g离心5分钟，去除上清，通过用PBS洗涤细胞，移除未结合的抗体。400g离心5分钟，去除PBS。每孔加入100μl 1:200稀释的PE缀合的抗人Fc抗体(SOUTHERN BIOTECH)，冰上避光孵育30分钟。400g离心5分钟，去除上清。通过用PBS洗涤细胞，移除未结合的PE缀合的抗人Fc抗体。用100μl PBS重悬细胞，通过FACS检测抗体与细胞的结合。结果见图3。
由图3可见，本发明的双特异性抗体Bi-119-112LC能够与细胞表面表达的PD-L1相结合，结合EC50为2.654nM，与亲本抗PD-L1抗体对细胞表面表达的PD-L1的结合能力(EC50为1.940nM)相似。
同样地，通过将携带克隆至多克隆位点MCS的人OX40 cDNA(Sino Biological Inc.)的pCHO1.0载体(Invitrogen)转染至中国仓鼠卵巢癌细胞(CHO)(Invitrogen)，产生过量表达人OX40的CHO细胞(CHO-OX40细胞)。
对CHO-OX40实施FACS检测，除了使用的细胞不同和使用ADI-20112抗体作为对照抗体之外，其余实验操作均与上述CHO-PD-L1细胞的FACS检测一样。
结果如图4所示。由图4可见，本发明的双特异性抗体Bi-119-112LC能够与细胞表面表达的OX40相结合，结合EC50为3.195nM，与亲本抗OX40抗体对细胞表面表达的OX40的结合能力(EC50为2.193nM)相似。
实施例1.5.本发明的抗OX40/PD-L1双特异性抗体同时结合过量表达OX40的CHO细胞和过量表达PD-L1的CHO细胞的分析-
为了验证本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC是否可以同时与来自不同细胞上的靶抗原结合，本实施例利用流式细胞技术，检测了所述双特异性抗体诱导的不同细胞交联情况。具体实验过程如下。
1)如实施例1.4所述获得CHO-PD-L1细胞和CHO-OX40细胞并培养。分别将含有CHO-PD-L1细胞和CHO-OX40细胞的培养物在离心机上以400g离心5分钟，去除细胞培养基。用PBS洗一遍后，再用PBS重悬细胞。计数细胞，并调整细胞密度为2×10 6个细胞/ml。将CHO-PD-L1细胞和CHO-OX40细胞分别按照1:5000加入CellTracker TM Deep Red(Thermo)和Cell Trace CFSE(Invitrogen)染料,于37℃放置30分钟。在离心机上以400g离心5分钟，去除上清液，用PBS洗一次细胞。
2)将梯度稀释的样品(抗OX40/PD-L1双特异性抗体Bi-119-112LC、抗PD-L1人源化Nb-Fc抗体(PCT/CN2017/095884)、和抗OX40抗体ADI-20112(中国发明专利申请号201710185400.8))分别加入U型底96孔板中。加入上述1)的染色后CHO-PD-L1细胞，混合(细胞终密度为1.5×10 6个/ml)。将U型底96孔板4℃放置30分钟后取出板，于400g离心5分钟，然后用PBS洗四次，并用PBS重悬细胞。
3)向U型底96孔板的上述2)的细胞悬液中加入上述1)的染色后的CHO-OX40细胞，使得CHO-OX40细胞终密度为1×10 6个/ml，于室温放置1小时后进行FACS检测。通道2及通道4双阳性细胞的比例可反映出由抗OX40/PD-L1双特异性抗体Bi-119-112LC引起的细胞交联情况。
FACS检测结果如图5所示，抗OX40/PD-L1双特异性抗体Bi-119-112LC能够诱导CHO-PD-L1细胞和CHO-OX40细胞的交联，由此表明本发明的双特异性抗体能够同时结合来自不同细胞表面的靶抗原。本实施例中使用的IgG1阴性对照的重链(HC)氨基酸序列如SEQ ID NO:29所示；IgG1阴性对照的轻链(LC)氨基酸序列如SEQ ID NO:30所示。
实施例1.6.本发明的抗OX40/PD-L1双特异性抗体阻断PD-1与过量表达PD-L1的CHO细胞的结合分析
为了验证本发明抗OX40/PD-L1双特异性抗体是否可以阻断PD-1与过量表达PD-L1的CHO细胞结合，本实施例利用流式细胞技术，检测了本发明的抗OX40/PD-L1双特异性抗体阻断PD-1蛋白与过量表达PD-L1的CHO细胞结合，详细实验过程如下：
1)如实施例1.4所述获得CHO-PD-L1细胞并培养。将含有2.4×10 7个CHO-PD-L1细胞的培养物在离心机上以400g离心5分钟，去除细胞培养基。用PBS洗一遍后，再用5ml PBS重悬细胞。
2)细胞铺板：将1)中处理好的CHO-PD-L1细胞每孔50μl加入到96孔U底血凝板中备用。
3)梯度浓度样品溶液配制：向5ml PBS中加入生物素化的人PD-1蛋白(AcroBiosystems,PD1-H82F2)200μl(人PD-1蛋白浓度0.2mg/ml)，混匀。用生物素化的人PD-1和PBS混合液将待测样品稀释为：以1000nM为起始浓度，后面11个浓度点3倍稀释，共12个浓度点。
4)将配置好的梯度浓度样品每孔50μl加入到2)中铺好细胞的96孔U底血凝板中，混匀，4℃，孵育30分钟，400g，5min，离心，去除上清，每孔加入150μl PBS，400g，5min，离心，去除上清，反复重复三次。
5)每孔加入1:200稀释的Streptavidin-R-phycoerythrin(SAPE)(THERMO，S21388)100μl，4℃，30min。
6)每孔加入150μl PBS，400g，5min，离心，去除上清，反复重复两次，用100μl PBS重悬，流式细胞仪(BD Biosciences,ACCURIC6)检测。
本实施例中使用的IgG1阴性对照同上述实施例1.5中使用的IgG1阴性对照。实验结果如图 6所示，本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC可以有效阻断PD-1与过量表达PD-L1的CHO细胞的结合，且阻断活性与抗PD-L1人源化Nb-Fc抗体相似(抗OX40/PD-L1双特异性抗体的IC50为3.522nM，抗PD-L1人源化Nb-Fc抗体的IC50为4.906nM)。
实施例1.7.基于萤光素酶报告基因检测抗OX40/PD-L1双特异性抗体的抗PD-L1活性
为了确定抗OX40/PD-L1双特异性抗体是否可以解除PD-1/PD-L1相互作用对NFAT信号通路的抑制作用，本实施例使用萤光素酶报告基因检测细胞株(Promega,CS187109)，通过检测萤光素酶的表达反应出双特异性抗体对PD-1/PD-L1相互作用的抑制能力，详细实验过程如下：
考虑到对抗体的探索应该建立在对其作用机制(mechanisms of action；MOA)的了解和生物学活性的基础上，本实施例使用PD-1/PD-L1 Blockade Bioassay,Cell Propagation Model(Promega公司)，研究了本发明的双特异性抗体的抗PD-L1生物学活性。
Promega公司的PD-1/PD-L1Blockade Bioassay是一种生物学相关的基于MOA的测定法，用于测定能够阻断PD-1/PD-L1相互作用的抗体的效力和稳定性。该测定法由两种基因工程细胞系组成：
·PD-1效应细胞：稳定表达人PD-1和由活化的T细胞的核因子(nuclear factor of activated T cells；NFAT)诱导表达萤光素酶的Jurkat T细胞。
·PD-L1 aAPC/CHO-K1细胞：稳定表达人PD-L1的CHO-K1细胞和以抗原非依赖性方式活化相应TCR的细胞表面蛋白。
PD-1与PD-L1结合可以阻断NFAT下游信号的转导，从而抑制萤光素酶的表达，当加入PD-1抗体或者PD-L1抗体时，这种阻断效应被反转，萤光素酶表达，从而检测到荧光信号。该检测法灵敏度、特异性、准确度都很好，且稳定性很好。
根据制造商的产品说明书进行检测。
1)活性检测前一天铺PD-L1 aAPC/CHO-K1细胞：弃培养上清，PBS清洗一次，加入胰酶(Gibco,25200072)，37℃，孵育3-5min，用四倍体积的含10％FBS(HyClone,SH30084.03)的RPMI1640(Gibco,22400-071)培养基终止消化，收集细胞，取少量细胞混合液测定细胞浓度，取所需体积的细胞液，400g，离心10min，弃上清，用含10％FBS(HyClone,SH30084.03)的RPMI1640(Gibco,22400-071)培养基作为测定缓冲液(assay buffer)重悬细胞，使得细胞密度为4×10 5个细胞/ml。将细胞悬液加入96孔白色细胞培养板(Nunclon，136101)100μL/孔，96孔白色细胞培养板的边孔加入PBS，200μl/孔。细胞 于二氧化碳培养箱中37℃，5％CO 2培养箱中培养过夜。
2)取无菌96孔板(Nunclon，442404)，用含10％FBS的RPMI1640培养基将待测样品稀释为：以200nM为起始浓度，第二个浓度点2至第12浓度点3倍稀释，共12个浓度点。
3)取PD-1效应细胞，计数，400g，离心5min，用assay buffer重悬细胞，使得细胞浓度为1.25×10 6cells/ml。
4)从培养箱中取出白色细胞培养板，弃95μl/孔，依次加入2)中稀释好的抗体40μl以及3)中处理好的细胞，每孔40μl Jurkat/PD-1细胞。
5)二氧化碳培养箱中37℃，5％CO 2培养条件下培养6小时。
6)取出白色细胞培养板，室温静置5-10min。
7)将Bio-Glo TM缓冲液(Promega，G7940)融化，加入Bio-Glo TM底物(Promega，G7940)，混匀。将所获得的Bio-Glo TM试剂以80μl/孔加入上述培养6小时后的检测板的孔中。室温放置5至10分钟。
8)用Spectra Max I3酶标仪(Thermo，Max i3)，收集全波长化学发光，每孔收集时间为1000ms。
本实施例中使用的IgG1阴性对照同上述实施例1.5中使用的IgG1阴性对照。实验结果如图7所示，本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC可以有效解除PD1/PD-L1相互作用对NFAT信号通路的阻断效应，且活性与抗PD-L1人源化Nb-Fc抗体相似(抗OX40/PD-L1双特异性抗体的EC50为0.4585nM，抗PD-L1人源化Nb-Fc抗体的EC50为0.3283nM)。
实施例1.8.基于萤光素酶报告基因检测本发明的抗OX40/PD-L1双特异性抗体介导PD-L1依赖的激活OX40介导的信号通路的检测
为了检测本发明的抗OX40/PD-L1双特异性抗体在实施例1.4所述获得CHO-PD-L1细胞存在的情况下，激活OX40介导的信号通路生物活性。本实施例使用信达生物制药(苏州)有限公司的Jurkat-OX40-NFkB-Luc-Rep稳定细胞株，测量OX40介导的转录活化来评估本发明的抗OX40/PD-L1双特异性抗体是否具有抗OX40抗体的激活剂活性。用抗人CD3(BD Biosciences,目录号:555329)、抗人CD28(BD Biosciences,目录号:555725)加上溶液中的本发明的抗体来活化导入了人OX40构建体(购自Sino)和NFkB-萤光素酶构建体(NFkB启动子-luc，Promega)并过量表达人OX40的Jurkat细胞(获自美国ATCC)持续16小时，然后加入Bio-Glo TM试剂显色。具体实验过程如下：
溶液配制：分析缓冲液：RPIM-1640(90％)(Gibco，22400-071)，FBS(10％)(HyClone，SH30084.03)，抗人CD3(2μg/ml)(BD Biosciences,目录号:555329)，抗人CD28(2μg/ml)(BD Biosciences,目录号:555725)，现配现用。
实验步骤：
1)取少量细胞悬液，用细胞计数板测定细胞密度，400g，离心10min，去掉上清，使用分析缓冲液温和的重悬细胞，Jurkat-OX40-NFkB-Luc-Rep细胞密度为4×10 5个/ml； CHO-PD-L1细胞密度为4×10 5个/ml。
2)将细胞悬液移至加样槽中，取96孔白色细胞培养板(NUNC，目录号：136101)。每孔加入1)中处理好的50μL Jurkat-OX40-NFkB-Luc-Rep细胞和50μL CHO-PD-L1细胞悬液，加入待测样品，样品的起始浓度为100nM，第二个浓度点2至第13浓度点3倍稀释，共13个浓度点，一式三份重复。
3)二氧化碳培养箱中37℃，5％CO 2培养条件下培养16小时。
4)将Bio-Glo TM缓冲液(Promega，目录号:G7940)融化，加入Bio-Glo TM底物(Promega，目录号:G7940)，混匀。将所获得的Bio-Glo TM试剂以80μl/孔加入上述培养16小时后的检测板的孔中。室温放置5至10分钟，用Spectra Max I3酶标仪(Thermo，Max i3)，收集全波长化学发光，每孔收集时间为1000ms。
本实施例中使用的IgG1阴性对照同上述实施例1.5中使用的IgG1阴性对照。实验结果如图 8所示，在有PD-L1表达的细胞体系中，本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC具有显著的NFkB信号通路的激活作用，而抗OX40抗体ADI-20112则检测到较低的NFkB信号通路激活效应，抗PD-L1人源化Nb-Fc抗体没有NFkB信号通路激活效应。本发明的抗OX40/PD-L1双特异性抗体显示出了在PD-L1表达的细胞存在的情况下，能够更好的激活OX40下游的NFkB信号通路。
实施例1.9.本发明的抗OX40/PD-L1双特异性抗体的热稳定性检测
差示扫描荧光法(differential scanning fluorimetry；DSF)能够根据蛋白质图谱中的荧光变化过程提供有关蛋白质结构稳定性的信息，检测蛋白的构型变化，获得蛋白质的熔解温度(T m)。本实施例采用DSF法测定了本发明抗OX40/PD-L1双特异性抗体的T m值。
将本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC抗体分别用PBS溶液稀释至1mg/ml。
向4μl SYPRO Orange Protein Gel Stain(Gibco，目录号：S6650)中加入196μl PBS，将SYPRO Orange Protein Gel Stain稀释50倍。
向96孔PCR板(Nunc)中加入50μl的上述浓度为1mg/ml的双特异性抗体，并加入10μl的上述50倍稀释的SYPRO Orange Protein Gel Stain，然后加入40μl水。置于7500 Real Time PCR系统(Applied Biosystems,AB/7500)进行检测。设置系统温度为每分钟升高0.5度，荧光曲线绝对值出现峰值时对应的温度即为该蛋白质的T m。
实验结果如下表3和图 9所示。本发明的双特异性抗体T m>60℃，因此，具有较好的热稳定性。
表3.双特异性抗体T m值测定结果
Figure PCTCN2019079671-appb-000007
实施例1.10本发明的抗OX40/PD-L1双特异性抗体的热稳定性检测
为了进一步确认双特异性抗体的稳定性，本实施例通过检测制备的一批抗体在40℃ 放置0、1、3、7、10、20、30天之后的纯度的变化，从而评价了抗体的长期热稳定性。经SEC检测，所述制备的一批Bi-119-112LC抗体的初始纯度为92.91％。实验方法如下：浓缩抗体样品至5mg/ml(溶于PBS中)，分装于EP管中，200μl/管，避光置于40℃。在第0、1、3、7、10、20、30天各取一管利用SEC-HPLC测定其单体主峰纯度。
实验结果如表4所示。本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC在40℃放置30天，其单体主峰比例降低幅度仅为3.69％。结果表明，本发明的抗OX40/PD-L1双特异性抗体具有较好的热稳定性。
表4.双特异性抗体40℃培养时单体主峰比例变化
放置于40℃(天) Bi-119-112LC
0 92.91％
1 91.51％
3 91.10％
7 90.93％
10 90.45％
20 89.72％
30 89.22％
实施例1.11.本发明的抗OX40/PD-L1双特异性抗体对人CD4 +T细胞的激活作用检测
本实施例检测了在体外抗OX40/PD-L1双特异性抗体对人CD4 +T细胞的激活作用，详细实验过程如下：
复苏人的PBMC细胞(ALLCELLS，PB005F)，静置3小时贴壁后的细胞即为单核细胞，添加10ml AIM
Figure PCTCN2019079671-appb-000008
Medium CTS(GIBCO，A3021002)培养基，加入IL4(20ng/ml)(R&D，204-IL)，GM-CSF(10ng/ml)(R&D，215-GM)诱导单核细胞分化为树突状细胞(即，DC细胞)，培养至第5天，添加诱导DC成熟的细胞因子TNFα(1000U/ml)(R&D，目录号:210-TA)，RhIL-1β(5ng/ml)(R&D，目录号:201-LB)，RhIL-6(10ng/ml)(R&D，目录号:206-IL)，1μM PGE(Tocris，目录号:2296)，二氧化碳培养箱中37℃，5％CO 2培养条件下继续培养2天，作为淋巴细胞混合反应(MLR)的成熟DC细胞(moDC)；
复苏人的PBMC细胞(ALLCELLS，目录号:PB005F)，按照人CD4 +T细胞富集试剂盒(STEMCELL，目录号:19052)的说明书，实施CD4 +T细胞分离。简而言之，上述将PBMC静置培养2小时后吸取的悬浮细胞液置于20ml离心管中，300g离心10分钟，向细胞沉淀物中加入500μl分离液和100μl试剂盒中配备的纯化抗体，4℃孵育20分钟，用分离液清洗一次，再加入500μl珠缓冲液孵育15分钟，磁场去除珠，用AIM
Figure PCTCN2019079671-appb-000009
Medium CTS(GIBCO，目录号:A3021002)培养基洗一次，使用8ml AIM
Figure PCTCN2019079671-appb-000010
Medium CTS培养基培养获得的CD4 +T细胞。按照CD4 +T细胞：抗CD3/CD28珠＝1:1加入Dynabeads  Human T-Activator CD3/CD28(INVITROGEN，目录号:11131D)中，二氧化碳培养箱中37℃，5％CO 2培养条件下培养3天，对CD4 +T细胞实施珠刺激；
将上述分离的DC细胞与经珠刺激的CD4 +T细胞混合，加入葡萄球菌肠毒素E超抗原(Toxin technology，目录号:ET404)，终浓度1ng/ml，每孔体积200μl，DC细胞12000个，CD4 +T细胞120000个，加入梯度稀释的抗体，混合培养3天，使用Cisbio IL2检测试剂盒(CISBIO，目录号:62HIL02PEG)检测每个样品中的IL2表达量，不同抗体IL2表达量反应了该抗体对T细胞的激活能力。
结果如图10所示，本发明的抗OX40/PD-L1双特异性抗体Bi-119-112LC可以在体外有效激活人CD4 +T细胞，其激活效果比抗PD-L1人源化Nb-Fc抗体、抗OX40抗体ADI-20112更强。
实施例2.抗VEGF/GITR双特异性抗体的构建、表达、纯化及性质鉴定
实施例2.1.抗VEGF/GITR双特异性抗体的构建
在本实施例中，构建了2种结构的抗VEGF/GITR双特异性抗体，分别命名为(1)双特异性抗体Bi-2-50，其结构示意图如图11A所示；和(2)双特异性抗体Bi-2-51，其结构示意图如图11B所示。下面对这两种抗VEGF/GITR双特异性抗体分别进行描述。
(1)从图11A的结构示意图可见，双特异性抗体Bi-2-50由左右对称的4条多肽链组成，其中左半部分的2条多肽链(即，肽链#1和肽链#2)从N端至C端分别具有SEQ ID NO:18和SEQ ID NO:21所示的氨基酸序列。具体而言，在SEQ ID NO:18所示的肽链#1中包含衍生自抗VEGF抗体Avastin的SEQ ID NO:20所示的VL氨基酸序列和在所述VL氨基酸序列C端的SEQ ID NO:8所示的人κ轻链恒定区(CL)氨基酸序列；在SEQ ID NO:21所示的肽链#2中包含衍生自抗VEGF单克隆抗体Avastin的SEQ ID NO:22所示的VH氨基酸序列、在所述VH氨基酸序列C端的衍生自人IgG1的SEQ ID NO:23所示的CH1氨基酸序列、在所述CH1氨基酸序列C端的SEQ ID NO:9所示的连接肽氨基酸序列和SEQ ID NO:24所示的抗GITR VHH氨基酸序列、以及SEQ ID NO:13所示的衍生自人IgG1的Fc区氨基酸序列。
(2)从图11B的结构示意图可见，双特异性抗体Bi-2-51由左右对称的4条多肽链组成，其中左半部分的2条多肽链(即，肽链#1和肽链#2)从N端至C端分别具有SEQ ID NO:18和SEQ ID NO:28所示的氨基酸序列。在SEQ ID NO:28所示的肽链#2中从N端至C端包含SEQ ID NO:22所示的衍生自抗VEGF单克隆抗体Avastin的VH氨基酸序列、SEQ ID NO:23所示的衍生自人IgG1的CH1氨基酸序列、SEQ ID NO:24所示的抗GITR VHH氨基酸序列、和SEQ ID NO:13所示的衍生自人IgG1Fc区的氨基酸序列。
实施例2.2.抗VEGF/GITR双特异性抗体的表达、纯化和分析
在本实施例中，将编码实施例2.1中构建的抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51的肽链#1、肽链#2的核苷酸序列分别通过多克隆位点连接入市售的真核表达载体pTT5，在真核细胞中进行表达和纯化，获得了抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51。
质粒转染、抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51的表达和纯化操作同上述实施例1.2。双特异性抗体Bi-2-50和Bi-2-51的SEC结果分别如图12A和图12B所示。
经过纯化后，抗VEGF/GITR双特异性抗体均具有很好的纯度，双特异性抗体Bi-2-50和Bi-2-51的单体主峰纯度分别为99.57％和99.48％。
实施例2.3.测定抗VEGF/GITR双特异性抗体的解离常数
使用Octet系统(ForteBio公司生产)通过动力学结合测定法确定本发明的上述示例性抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51结合VEGF和GITR的平衡解离常数(K D)。除了使用的抗体和抗原不同之外，其他具体实验过程同上述实施例1.3。检测结果见下表5和表6。
作为针对GITR的亲本单特异性抗体，使用了名为“hcIgG-10”的具有SEQ ID NO:31所示的氨基酸序列的抗体，其从N端至C端包含SEQ ID NO:24所示的抗GITR VHH氨基酸序列、“DKTHT”肽段和SEQ ID NO:13所示的衍生自人IgG1的Fc区氨基酸序列。
表5.通过ForteBio动力学结合测定法确定的抗VEGF/GITR双特异性抗体对GITR的亲和力
Figure PCTCN2019079671-appb-000011
表6.通过ForteBio动力学结合测定法确定的抗VEGF/GITR双特异性抗体对VEGF165的亲和力
Figure PCTCN2019079671-appb-000012
通过表5和表6中的数据可见，本发明的抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51均能够同时与溶液中的VEGF165(R&D，293-VE-500)和GITR(AcroBiosystems，GIR-H5228-1MG)蛋白结合，且维持了亲本抗体Avastin或hcIgG-10的亲和力常数。
实施例2.4.本发明的抗VEGF/GITR双特异性抗体与过量表达VEGF或GITR的CHO细胞的结合分析
通过FACS测量了本发明的抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51与过量表达VEGF或GITR的CHO细胞的结合。除了使用的抗体和抗原不同之外，其他具体实 验操作同上述实施例1.4。本实施例中使用的IgG1阴性对照同上述实施例1.5中使用的IgG1阴性对照。结果如图13所示。
由图13可见，本发明的抗VEGF/GITR双特异性抗体Bi-2-50和Bi-2-51均能够与细胞表面表达的GITR结合，结合EC50分别为2.990nM和3.168nM。亲本抗体hcIgG-10与细胞表面GITR结合的EC50为0.6061nM。
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。在这个方面，本发明范围仅由以下权利要求限定。

Claims (24)

  1. 一种抗体分子，其包含(i)单结构域抗原结合位点；(ii)结合抗原的Fab片段；其中所述(i)位于所述(ii)的轻链可变结构域(VL)的N端或轻链恒定区(CL)的C端，或者所述(i)位于所述(ii)的重链可变结构域(VH)的N端或免疫球蛋白CH1结构域的C端，且所述(i)和(ii)分别结合相同或者不同的抗原，所述(i)和(ii)之间具有或者不具有连接肽；以及位于所述(i)和(ii)的C端的(iii)免疫球蛋白Fc结构域。
  2. 根据权利要求1所述的抗体分子，其中所述单结构域抗原结合位点选自重链可变结构域(VH)、轻链可变结构域(VL)、天然缺乏轻链的抗体的重链可变结构域(例如，骆驼科物种中天然存在的重链抗体的重链可变结构域)、鱼类中称为新型抗原受体(NAR)的免疫球蛋白(如鲨鱼血清中天然存在的IgNAR)中的VH样单结构域、和衍生自它们的经重组的单结构域抗原结合位点(例如，骆驼化的人VH结构域、人源化的骆驼科抗体重链可变结构域)，例如，所述单结构域抗原结合位点选自骆驼科物种中天然存在的重链抗体的重链可变结构域、骆驼化的人VH结构域和人源化的骆驼科抗体重链可变结构域(它们简称为“VHH”)。
  3. 根据权利要求1或2所述的抗体分子，其中所述免疫球蛋白是IgG1、IgG2或IgG4免疫球蛋白，优选地，所述免疫球蛋白是IgG1免疫球蛋白，更优选地，所述免疫球蛋白是人IgG1免疫球蛋白；所述免疫球蛋白的轻链型别是κ型或λ型，优选地为κ型。
  4. 根据权利要求1－3中任一项所述的抗体分子，其中所述Fc结构域包含免疫球蛋白恒定部分的铰链区，并且所述抗体分子的重链彼此通过所述铰链区处的二硫键稳定缔合，例如，在所述抗体分子的重链的Fc结构域中包含具有“CPPC”氨基酸残基的铰链区，从而所述重链彼此通过所述铰链区处氨基酸残基之间形成的二硫键稳定缔合；
    优选地，所述抗体分子的重链在各自的Fc结构域中还分别包含Y349C和S354C或者S354C和Y349C(根据Kabat的“EU编号”)，从而所述抗体分子的重链彼此在Fc区进一步形成链间二硫键。
  5. 根据权利要求1－4中任一项所述的抗体分子，其中所述Fc结构域中还包含影响抗体效应子功能的突变，例如，LALA突变。
  6. 根据权利要求1－5中任一项所述的抗体分子，其中所述抗体分子的重链在各自的Fc结构域中分别包含凸起或空穴，并且一条重链Fc结构域中的所述凸起或空穴可分别置于另一条重链Fc结构域中的所述空穴或凸起中，由此所述抗体分子的重链彼此形成“结入扣”的稳定缔合。
  7. 根据权利要求1－6中任一项所述的抗体分子，其中所述免疫球蛋白CH1结构域和轻链恒定结构域(CL)中分别包含凸起或空穴，并且CH1结构域中的所述凸起或空穴可分别置于CL结构域中的所述空穴或凸起中，由此所述抗体分子的重链和轻链彼此形成“结入扣”的稳定缔合。
  8. 根据权利要求1－7中任一项所述的抗体分子，其中所述(i)和(ii)分别结合相同抗原上的表位或者不同抗原上的表位，
    例如，所述(i)结合第一抗原的表位，所述(ii)结合第二抗原上的表位，由此，所述抗体分子是针对第一抗原和第二抗原的双特异性抗体。
  9. 根据权利要求1－8中任一项所述的抗体分子，其中所述连接肽是单独或组合使用的甘氨酸和/或丝氨酸残基，例如，所述连接肽包含氨基酸序列(Gly 4Ser)n，其中n是等于或大于1的正整数，例如，n是1－7中的正整数，例如，n是2。
  10. 根据权利要求1－9中任一项所述的抗体分子，其中所述抗原是细胞因子、生长因子、激素、信号传导蛋白、炎性介质、配体、细胞表面受体或其片段。
  11. 根据权利要求10所述的抗体分子，其中所述抗原选自肿瘤相关抗原、免疫检查点分子、血管新生诱导因子、肿瘤坏死因子受体超家族成员和免疫系统中的共刺激分子，以及这些分子的配体和/或受体。
  12. 根据权利要求11所述的抗体分子，其中所述抗原选自OX40、CD47、PD1、PD-L1、PD-L2、LAG-3、4-1BB(CD137)、VEGF和GITR。
  13. 根据权利要求1－12中任一项所述的抗体分子，其是包含四条多肽链的抗体分子，其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链以及位于所述免疫球蛋白轻链可变结构域(VL)的N端的单结构域抗原结合位点，例如VHH；第二多肽链和第四多肽链中的每一多肽链包含免疫球蛋白重链；或者
    其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链以及位于所述免疫球蛋白轻链恒定区(CL)的C端的单结构域抗原结合位点，例如VHH；第二多肽链和第四多肽链中的每一多肽链包含免疫球蛋白重链；或者
    其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链；第二多肽链和第四多肽链中的每一多肽链包含免疫球蛋白重链以及位于所述免疫球蛋白重链N端的单结构域抗原结合位点，例如VHH；或者
    其中第一多肽链和第三多肽链中的每一多肽链包含免疫球蛋白轻链；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变区、免疫球蛋白CH1结构域、单结构域抗原结合位点(例如VHH)、免疫球蛋白CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白重链可变结构域(VH)和免疫球蛋白轻链恒定区(CL)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1、CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)和单结构域抗原结合位点(例如VHH)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1、CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)和免疫球蛋白轻链恒定区(CL)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1、CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)和免疫球蛋白轻链恒定区(CL)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1结构域、单结构域抗原结合位点(例如VHH)、免疫球蛋白CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白轻链可变结构域(VL)和免疫球蛋白CH1结构域；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、免疫球蛋白CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)、免疫球蛋白CH1结构域和单结构域抗原结合位点(例如VHH)；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、免疫球蛋白CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)和免疫球蛋白CH1结构域；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含单结构域抗原结合位点(例如VHH)、免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、免疫球蛋白CH2、CH3和任选地CH4结构域；或者
    其中第一多肽链和第三多肽链中的每一多肽链从N端至C端包含免疫球蛋白轻链可变结构域(VL)和免疫球蛋白CH1结构域；第二多肽链和第四多肽链中的每一多肽链从N端至C端包含免疫球蛋白重链可变结构域(VH)、免疫球蛋白轻链恒定区(CL)、单结构域抗原结合位点(例如VHH)、免疫球蛋白CH2、CH3和任选地CH4结构域。
  14. 根据权利要求1－13中任一项所述的抗体分子，其是抗OX40/PD-L1双特异性抗体，且(i)单结构域抗原结合位点和(ii)结合抗原的Fab片段分别结合OX40或PD-L1分子，
    例如，所述抗体分子由左右基本上对称的4条多肽链组成，其中在左半部分的2条多肽链和右半部分的2条多肽链中，均包含(i)特异性结合PD-L1的单结构域抗原结合位点；(ii)特异性结合OX40的Fab片段；
    优选地，所述(i)单结构域抗原结合位点包含SEQ ID NO:3所示的CDR1、SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；所述(ii)结合抗原的Fab片段包含衍生自抗OX40抗体的SEQ ID NO:11/7所示的成对重链可变区序列/轻链可变区序列中的全部6个重链互补决定区(CDR)与轻链CDR，或者与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；
    更优选地，所述(i)单结构域抗原结合位点包含衍生自SEQ ID NO:1或SEQ ID NO:2所示的抗PD-L1VHH氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列；所述(ii)结合抗原的Fab片段包含衍生自抗OX40抗体的SEQ ID NO:11/7所示的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；
    最优选地，所述抗体分子的左半部分的2条多肽链分别包含SEQ ID NO:6所示的第一多肽链和SEQ ID NO:10所示的第二多肽链；分别包含SEQ ID NO:14所示的第一多肽链和SEQ ID NO:10所示的第二多肽链；分别包含SEQ ID NO:15所示的第一多肽链和SEQ ID NO:16所示的第二多肽链；分别包含SEQ ID NO:15所示的第一多肽链和SEQ ID NO:17所示的第二多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列；相应地，其中所述抗体分子的右半部分的2条多肽链分别包含SEQ ID NO:6所示的第三多肽链和SEQ ID NO:10所示的第四多肽链；分别包含SEQ ID NO:14所示的第三多肽链和SEQ ID NO:10所示的第四多肽链；分别包含SEQ ID NO:15所示的第三多肽链和SEQ ID NO:16所示的第四多肽链；分别包含SEQ ID NO:15所示的第三多肽链和SEQ ID NO:17所示的第四多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
  15. 根据权利要求1－13中任一项所述的抗体分子，其是抗VEGF/GITR双特异性抗体，且(i)单结构域抗原结合位点和(ii)结合抗原的Fab片段分别结合VEGF或GITR分子，
    例如，所述抗体分子由左右基本上对称的4条多肽链组成，其中在左半部分的2条多肽链和右半部分的2条多肽链中，均包含(i)特异性结合GITR的单结构域抗原结合位点；(ii)特异性结合VEGF的Fab片段；
    优选地，所述(i)单结构域抗原结合位点包含SEQ ID NO:25所示的CDR1、SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3，或者与所述3个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；所述(ii)结合抗原的Fab片段包含衍生自抗VEGF抗体的SEQ ID NO:22/20所示的成对重链可变区序列/轻链可变区序列中的全部6个重链互补决定区(CDR)与轻链CDR，或者与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；
    更优选地，所述(i)单结构域抗原结合位点包含衍生自SEQ ID NO:24所示的抗GITR VHH氨基酸序列，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列；所述(ii)结合抗原的Fab片段包含衍生自抗VEGF抗体的SEQ ID NO:22/20所示的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；
    最优选地，所述抗体分子的左半部分的2条多肽链分别包含SEQ ID NO:18所示的第一多肽链和SEQ ID NO:21所示的第二多肽链；分别包含SEQ ID NO:18所示的第一多肽链和SEQ ID NO:28所示的第二多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列；相应地，其中所述抗体分子的右半部分的2条多肽链分别包含SEQ ID NO:18所示的第三多肽链和SEQ ID NO:21所示的第四多肽链；分别包含SEQ ID NO:18所示的第三多肽链和SEQ ID NO:28所示的第四多肽链；或与任一所述序列基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更高同一)的序列。
  16. 多核苷酸，其编码权利要求1－15中任一项所述的抗体分子中的任意一条或者多条多肽链。
  17. 载体，优选地表达载体，其包含权利要求16的多核苷酸。
  18. 宿主细胞，其包含权利要求16所述的多核苷酸或权利要求17所述的载体，例如，所述宿主细胞是哺乳动物细胞，优选地是CHO细胞、HEK293细胞；所述宿主细胞是原核细胞，优选地是大肠杆菌细胞。
  19. 用于生产权利要求1－15中任一项所述的抗体分子的方法，所述方法包括步骤(i)在适于表达所述抗体分子的条件下培养权利要求18所述的宿主细胞，和(ii)从所述宿主细胞或所述培养基回收所述抗体分子。
  20. 药物组合物，其包含权利要求1－15中任一项所述的抗体分子和可药用载体。
  21. 根据权利要求20所述的药物组合物，其还包含至少一种其他有效成分。
  22. 免疫缀合物，其包含权利要求1－15中任一项所述的抗体分子和与所述抗体分子缀合的一个或多个异源分子，优选地，所述一个或多个异源分子是细胞毒性剂。
  23. 根据权利要求1－15中任一项所述的抗体分子、权利要求20－21所述的药物组合物、和权利要求22所述的免疫缀合物的用途，用作在个体中治疗和/或预防疾病的药物或用作疾病的诊断工具，优选地，所述个体是哺乳动物，更优选地是人。
  24. 根据权利要求23所述的用途，用于自身免疫病、急性和慢性炎性疾病、感染性疾病(例如，慢性传染病或败血症)、肿瘤的治疗和/或预防或诊断。
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