[go: up one dir, main page]

WO2022002006A1 - Fab-HCAb结构的结合蛋白 - Google Patents

Fab-HCAb结构的结合蛋白 Download PDF

Info

Publication number
WO2022002006A1
WO2022002006A1 PCT/CN2021/102935 CN2021102935W WO2022002006A1 WO 2022002006 A1 WO2022002006 A1 WO 2022002006A1 CN 2021102935 W CN2021102935 W CN 2021102935W WO 2022002006 A1 WO2022002006 A1 WO 2022002006A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
protein
antibody
variable region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/102935
Other languages
English (en)
French (fr)
Inventor
何云
石磊
黄冰
钟琛
吕强
王明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harbour Biomed Shanghai Co Ltd
Original Assignee
Harbour Biomed Shanghai Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harbour Biomed Shanghai Co Ltd filed Critical Harbour Biomed Shanghai Co Ltd
Priority to CN202180040307.2A priority Critical patent/CN115667316A/zh
Priority to US18/013,530 priority patent/US20230287143A1/en
Publication of WO2022002006A1 publication Critical patent/WO2022002006A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the field of biomedicine, in particular to a binding protein with a Fab-HCAb structure and its preparation and application.
  • Antibodies are immunoglobulins (Immunoglobulin, Ig) that are produced by B cells under the stimulation of the immune system and can specifically bind to the corresponding antigen.
  • the basic structure of antibodies of most species is a tetrameric form in the "Y" shape, containing two identical heavy chains (H chains) and two identical light chains (L chains), also known as “H2L2" ".
  • the heavy chain includes the heavy chain variable region (VH) near the N-terminus and the heavy chain constant region (CH) near the C-terminus;
  • the light chain includes the light chain variable region (VL) near the N-terminus and the light chain constant region (CL) near the C-terminus. ).
  • the heavy chain constant region of IgG antibody has three domains, namely CH1, CH2 and CH3; there is also a hinge region between CH1 and CH2.
  • the variable region of an antibody is its main site for recognizing and binding antigen; the variable region domains VH and VL and the constant region domains CH1 and CL of the antibody together constitute an antigen-binding fragment (Fab).
  • CH2 and CH3 constitute a crystallizable fragment (fragment crystallizable, Fc), which is the main site that exerts the effector function of the antibody and affects the serum half-life of the antibody.
  • heavy-chain antibody (heavy-chain antibody, HCAb) lacking light chain naturally in camelid and shark sera.
  • HCAb heavy-chain antibody
  • heavy chain antibodies derived from Camelidae have no CH1 region between the variable region of the heavy chain and the hinge region except for the lack of light chains, and only contain a variable heavy chain (VHH) and two heavy chain regions.
  • VHH variable heavy chain
  • Chain constant domains CH2 and CH3; its basic structure is a heavy chain dimer.
  • the VHH fragment of the heavy chain antibody of camelid is different from the VH characteristics of conventional antibodies.
  • the VHH structure cloned and expressed alone has the same structural stability and binding activity with the original heavy chain antibody as the original heavy chain antibody.
  • the molecular weight is only about 13KDa, therefore, it is also called nanobody (Nanobody) or single-domain antibody (single-domain antibody).
  • Nanobody Nanobody
  • Single-domain antibody single-domain antibody
  • Heavy chain antibodies or their derived nanobodies have unique advantages in molecular imaging, diagnostic reagents, etc., but their non-human properties and potential immunogenicity risks limit their therapeutic uses and require further antibody engineering. (eg antibody humanization) to make it meet the requirements for clinical treatment.
  • transgenic mouse constructed a transgenic mouse in which both the mouse endogenous antibody heavy chain locus and light chain locus were knocked out or inactivated, making it unable to produce mouse antibodies;
  • the antibody heavy chain gene fragments (V, D, J fragments) are transferred into the mouse, and the mouse's own rearrangement and mutation mechanism are used to generate antibodies with human antibody gene sequences, and because there is no light chain, the generated antibodies are Human heavy chain antibody.
  • the transgenic mouse can utilize the process of introducing gene mutation and natural selection after VDJ rearrangement in vivo to select VDJ combinations and mutations that are beneficial to the solubility of VH, and effectively improve the solubility of VH. Therefore, the transgenic mouse can produce natural natural selection in vivo. Absent human heavy chain dimer structure.
  • the fully human heavy chain antibody obtained from the transgenic mouse and the fully human single domain antibody derived therefrom have broad application prospects.
  • Bispecific antibodies (bispecific antibodies) and multispecific antibodies (multispecific antibodies) are a kind of artificial antibodies with two or more different specific antigen binding sites prepared by protein engineering technology on the basis of natural monoclonal antibodies.
  • Natural monoclonal antibodies are monospecific, that is, they can only recognize and bind to one antigen; bispecific antibodies can bind two different antigens or different epitopes on the same antigen; and multispecific antibodies may recognize more antigens. This enables bispecific antibodies to achieve some mechanisms of action and functional effects that monospecific antibodies cannot achieve, which greatly expands the therapeutic application scenarios of bispecific antibodies.
  • bispecific antibodies With the rise of tumor immunity in recent years, bispecific antibodies have attracted more and more attention, technology and financial support, becoming one of the fastest growing areas in the therapeutic antibody market.
  • bispecific antibodies The structural design of bispecific antibodies is very important. Naturally occurring bivalent IgG antibodies are composed of two identical heavy chains and two identical light chains and contain two identical antigen-binding sites. Bispecific antibodies need to introduce two different antigen-binding sites through structural design using methods such as protein engineering technology, and the polypeptide chains of the resulting molecules are derived from two different heavy chains and two different light chains. Therefore, one of the main challenges in bispecific antibody development is the problem of chain mismatch, that is, how to obtain functional bispecifics with the correct chain combination from more than 10 different combinations of heavy and light chains. antibody. To address this problem, scientists have developed a variety of development strategies and technology platforms to improve the homogeneity and yield of desired target products by introducing different design features or functional properties.
  • Adopting a symmetric structure is a strategy for solving the mismatch problem of chains.
  • Most symmetric structures are designed using "2+2" structures, also known as “tetravalent bispecific symmetric structures". Since their antigen-binding domains may have different structures, orientations, and positions, these symmetrically structured molecules have large differences in molecular size and pharmacological properties. Symmetric structure still has the problem of light chain mismatch; AbbVie's DVD-Ig technology platform, EpimAb's FIT-Ig technology platform, WuXi Biologics' WuXiBody technology platform, etc.
  • the molecular weight of double antibody molecules produced by technologies such as FIT-Ig is about 250KDa, and the larger molecular size may affect its ability to endocytosis and tissue penetration; and The introduction of the scFv structure may bring about the impact of stability and solubility; and the double antibody molecules produced by many technology platforms have at least three different polypeptide chains, which increases the complexity of the molecule.
  • Heavy chain antibodies and their derived single domain antibodies have their unique advantages in the construction of bispecific or even multispecific antibodies.
  • the antigen-binding domain of heavy chain antibodies is only one-quarter the size of the Fab of conventional antibodies; and there is no light chain, avoiding the problem of light chain mismatches. Therefore, by using heavy chain antibodies and their derived single domain antibodies, bispecific or even multispecific antibodies with smaller molecular weights, fewer polypeptide chains and simpler structures can be constructed.
  • fully human heavy chain antibodies have more advantages in immunogenicity and druggability than camelid heavy chain antibodies.
  • the present invention provides a bispecific binding protein with "Fab-HCAb structure” and a preparation method thereof and application.
  • the "Fab-HCAb structure” has the characteristics of smaller molecular weight, fewer polypeptide chains, simple structure, etc., and also has Fc effector functions similar to IgG antibodies, excellent molecular stability and pharmaceutical properties.
  • one of the technical solutions of the present invention is to provide a binding protein containing at least two protein functional domains, wherein the binding protein includes a protein functional domain A and a protein functional domain B; the protein functional domain Region A and the protein functional region B target different antigens or different epitopes of the same antigen, wherein the protein functional region A is a Fab structure, and the protein functional region B is a VH structure; the binding protein also includes Fc homodimers (containing at least one Fc);
  • the number of the protein functional regions A is two, and the number of the protein functional regions B is two;
  • the binding protein is a symmetrical structure, and the symmetrical structure is a left-right symmetrical structure;
  • the binding protein is sequentially from the N-terminus to the C-terminus of protein functional region A, protein functional region B and Fc, wherein the protein functional region A and the protein functional region B are connected by a first connecting peptide (L1), and the The protein functional domain B is linked to the Fc through a second linking peptide (L2).
  • two of the protein functional regions B and the Fc form a dimer form of a symmetrical single-chain antibody, and are connected at the N-terminus of the dimer of the single-chain antibody
  • the protein functional domain A can be associated with the protein functional domain by its CH1 (for example, see Figure 1, structure (2)) or CL (for example, see Figure 1, structure (1)).
  • CH1 for example, see Figure 1, structure (2)
  • CL for example, see Figure 1, structure (1)
  • the binding protein can be a tetravalent binding protein, for example, the binding protein has a structure as shown in structure (1) or (2) in Figure 1; the binding protein has two different polypeptide chains .
  • the binding protein has four polypeptide chains, which are two identical short chains (or “polypeptide chain 1") and two identical long chains (or “polypeptide chain 2"), wherein ( 1) The short chain (or “polypeptide chain 1") sequentially includes VH_A-CH1 from the N-terminus to the C-terminus, and the long chain (or “polypeptide chain 2") sequentially includes VL_A- CL-L1-VH_B-L2-CH2-CH3; or (2) the short chain (or “polypeptide chain 1") sequentially includes VL_A-CL from the N-terminus to the C-terminus, and the long chain (or “polypeptide chain 1") sequentially includes VL_A-CL.
  • Chain 2 includes VH_A-CH1-L1-VH_B-L2-CH2-CH3 sequentially from N-terminus to C-terminus.
  • the C-terminus of the protein functional domain A is connected to the N-terminus of the protein functional domain B with the C-terminus of the CL, and the VL_A of the protein functional domain A and the VH_B of the protein functional domain B are fused on the same polypeptide chain , compared with structure (2), the mismatch by-products generated by the association of VL_A and VH_B can be avoided.
  • VL, VH, CL and CH are conventional in the art, and represent light chain variable region, heavy chain variable region, light chain constant region and heavy chain constant region respectively, wherein CH includes CH1, CH2 and CH3, are the first, second and third domains of the heavy chain constant region respectively; the CL is the light chain constant region domain; _A and _B respectively represent that the functional region is a protein functional region A or a protein functional region B or Its composition (i.e., VH_A represents the heavy chain variable region of protein functional region A, VH_B represents the heavy chain variable region of protein functional region B, and VL_A represents the light chain variable region of protein functional region A); "-" represents the link Polypeptide bonds in different structural regions are used to separate different structural regions; the C-terminus is the carboxyl terminus of the peptide chain (also can be written as "C'"), and the N-terminus is the amino terminus of the peptide chain (also written as "N'").
  • L1 and L2 can be the same sequence. In other embodiments, L1 and L2 may be different sequences.
  • the L1 and/or L2 are "-", the length of the linking peptide is 0.
  • the L1 (first linker peptide) and L2 (second linker peptide) independently may be, for example, "-", GS or as shown in the amino acid sequences of SEQ ID NOs: 161-182.
  • the length of the L1 may preferably be 0, or as shown in the amino acid sequence of SEQ ID NOs: 163, 164 or 167.
  • the L2 may preferably be as shown in the amino acid sequence of SEQ ID NOs: 169, 178 or 179.
  • the L1 and L2 are shown in the amino acid sequences of SEQ ID NO: 167 and SEQ ID NO: 179, respectively.
  • the L1 has a length of 0 and the L2 is shown in the amino acid sequence of SEQ ID NO:178.
  • the L1 has a length of 0 and the L2 is shown in the amino acid sequence of SEQ ID NO:179.
  • the L1 and L2 are shown in the amino acid sequences of SEQ ID NO: 163 and SEQ ID NO: 178, respectively.
  • the L1 and L2 are shown in the amino acid sequences of SEQ ID NO: 164 and SEQ ID NO: 178, respectively. In some embodiments, the L1 and L2 are shown in the amino acid sequences of SEQ ID NO: 167 and SEQ ID NO: 178, respectively. In some embodiments, the L1 and L2 are shown in the amino acid sequences of SEQ ID NO: 163 and SEQ ID NO: 169, respectively.
  • the protein functional region A is also referred to as the antibody A against the first antigen or the first antigen binding domain; the protein functional region B is also referred to as the antibody B against the second antigen or the first antigen binding domain. Two antigen-binding domains.
  • the bispecific binding protein of the "Fab-HCAb structure” contains at least one heavy chain variable region domain VH derived from a human heavy chain antibody, and is capable of binding two or more Multiple antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope.
  • the heavy chain constant region contained in the bispecific binding protein of the "Fab-HCAb structure" may be preferably the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4 or its Mutations; the mutations are preferably selected from one or more of C220S, N297A, L234A, L235A, G237A and P329G, the mutation sites using the EU numbering convention.
  • the heavy chain constant region may comprise one, two or three mutations of L234A, L235A, G237A, N297A or P329G, eg a combination of mutations comprising L234A and L235A (LALA) or a combination of mutations comprising L234A, L235A and P329G (AAG) or a mutational combination of L234A, L235A and G237A (AAA) and the like.
  • LALA combination of mutations comprising L234A and L235A
  • AAG combination of mutations comprising L234A, L235A and P329G
  • AAAA mutational combination of L234A, L235A and G237A
  • the antigen is selected from one or more of PD-L1, HER2, B7H4, CTLA4, OX40, 4-1BB, and BCMA.
  • the binding protein contains at least two protein functional regions, namely protein functional region A and protein functional region B; the protein functional region A and the protein functional region B are independently derived from PD-L1 antibody or its antigen-binding fragment, HER2 antibody or antigen-binding fragment thereof, B7H4 antibody or antigen-binding fragment thereof, CTLA4 antibody or antigen-binding fragment thereof, OX40 antibody or antigen-binding fragment thereof, 4-1BB antibody or antigen-binding fragment thereof, and BCMA antibody or antigen-binding fragment thereof one or more of.
  • the protein functional region A is a Fab derived from a PD-L1 antibody or an antigen-binding fragment thereof, a HER2 antibody or an antigen-binding fragment thereof, a B7H4 antibody or an antigen-binding fragment thereof, or a BCMA antibody or an antigen-binding fragment thereof
  • the protein functional region B is a VH derived from CTLA4 antibody or its antigen-binding fragment, 4-1BB antibody or its antigen-binding fragment, OX40 antibody or its antigen-binding fragment, or BCMA antibody or its antigen-binding fragment.
  • the protein functional region A is a Fab derived from a HER2 antibody or an antigen-binding fragment thereof, and the protein functional region B is a VH derived from a CTLA4 antibody or an antigen-binding fragment thereof; or , the protein functional region A is a Fab derived from a PD-L1 antibody or an antigen-binding fragment thereof, and the protein functional region B is a VH derived from a 4-1BB antibody or an antigen-binding fragment thereof; or, the protein functional region Region A is a Fab derived from a B7H4 antibody or an antigen-binding fragment thereof, and the protein functional region B is a VH derived from a 4-1BB antibody or an antigen-binding fragment thereof; or, the protein functional region A is derived from a B7H4 antibody or the Fab of an antigen-binding fragment thereof, and the protein functional region B is a VH derived from an OX40 antibody or an antigen-binding fragment thereof;
  • the PD-L1 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, amino acids
  • VL comprises LCDR1, LCDR2 and LCDR3, amino acids
  • the sequences are shown in SEQ ID NOs: 75, 85 and 97, respectively; the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 13, 32 and 54, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the B7H4 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively As shown in SEQ ID NOs: 78, 83 and 100; the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 15, 37 and 59, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, amino acids
  • VL comprises LCDR1, LCDR2 and LCDR3, amino acids
  • VH comprises HCDR1, HCDR2 and HCDR3
  • amino acid sequences are shown in SEQ ID NOs: 11, 30 and 52, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH); the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 14, 35 and 57 are shown. The amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • VH heavy chain variable region
  • the OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are as shown in SEQ ID NOs: 13, 36 and 1, respectively. 58 shown.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 17, 39 and 61 shown.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively As shown in SEQ ID NOs: 77, 87 and 99; the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 13, 34 and 56, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are as shown in SEQ ID NOs: 10, 29 and 1, respectively. 51 shown.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the HER2 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively As shown in SEQ ID NOs: 74, 84 and 96; the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 12, 31 and 53, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the PD-L1 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising as shown in SEQ ID NO: 118
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequence shown, the VH includes the amino acid sequence shown in SEQ ID NO: 108.
  • the B7H4 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising as shown in SEQ ID NO: 121
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequence, the VH includes the amino acid sequence shown in SEQ ID NO:113.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising as set forth in SEQ ID NO: 116
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequence shown, the VH includes the amino acid sequence shown in SEQ ID NO: 106.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 111.
  • VH heavy chain variable region
  • the OX40 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:112.
  • VH heavy chain variable region
  • the BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:115.
  • VH heavy chain variable region
  • the BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising as shown in SEQ ID NO: 120
  • VL light chain variable region
  • VH heavy chain variable region
  • the amino acid sequence, the VH includes the amino acid sequence shown in SEQ ID NO: 110.
  • the CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:105.
  • VH heavy chain variable region
  • the HER2 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising as shown in SEQ ID NO: 117
  • the amino acid sequence, the VH comprises the amino acid sequence shown in SEQ ID NO:107.
  • the PD-L1 antibody or antigen-binding fragment thereof comprises a light chain whose sequence is shown in SEQ ID NO: 136 and a heavy chain whose sequence is shown in SEQ ID NO: 126.
  • the B7H4 antibody or antigen-binding fragment thereof comprises a light chain with a sequence shown in SEQ ID NO:139 and a heavy chain with a sequence shown in SEQ ID NO:131.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a light chain whose sequence is shown in SEQ ID NO:134 and a heavy chain whose sequence is shown in SEQ ID NO:124.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain whose sequence is shown in SEQ ID NO: 129.
  • the OX40 antibody or antigen-binding fragment thereof comprises a heavy chain whose sequence is shown in SEQ ID NO: 130.
  • the BCMA antibody or antigen-binding fragment thereof comprises a heavy chain whose sequence is shown in SEQ ID NO: 133.
  • the BCMA antibody or antigen-binding fragment thereof comprises a light chain with the sequence shown in SEQ ID NO:138 and a heavy chain with the sequence shown in SEQ ID NO:128.
  • the CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain whose sequence is shown in SEQ ID NO: 123.
  • the HER2 antibody or antigen-binding fragment thereof comprises a light chain with a sequence shown in SEQ ID NO:135 and a heavy chain with a sequence shown in SEQ ID NO:125.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively as SEQ ID NOs: 75, 85 and 97
  • the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 13, 32 and 54, respectively
  • the protein functional region B comprises a heavy chain variable region (VH), the VH Comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences are shown in SEQ ID NOs: 14, 35 and 57, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively as SEQ ID NOs: 78, 83 and 100 shown
  • the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 15, 37 and 59, respectively
  • the protein functional region B comprises a heavy chain variable region (VH), the VH Comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences are shown in SEQ ID NOs: 14, 35 and 57, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively as SEQ ID NOs: 78, 83 and 100 shown
  • the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 15, 37 and 59, respectively
  • the protein functional region B comprises a heavy chain variable region (VH), the VH Comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences are shown in SEQ ID NOs: 13, 36 and 58, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively as SEQ ID NOs: 77, 87 and 99 shown; the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 13, 34 and 56, respectively; and, the protein functional region B comprises a heavy chain variable region (VH), the VH Comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences shown are as SEQ ID NOs: 17, 39 and 61, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region (VL) and a heavy chain variable region (VH)
  • the VL comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively as SEQ ID NOs: 74, 84 and 96 shown
  • the VH comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are shown in SEQ ID NOs: 12, 31 and 53, respectively
  • the protein functional region B comprises a heavy chain variable region (VH), the VH Comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences are shown in SEQ ID NOs: 10, 29 and 51, respectively.
  • the amino acid sequences of the listed CDRs are shown according to the Chothia definition rules.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 118 and a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 108;
  • the protein functional region B comprises The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 111.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 121 and a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 113;
  • the protein functional region B comprises The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 111.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 121 and a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 113;
  • the protein functional region B comprises The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 112.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 120 and a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 110;
  • the protein functional region B comprises The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 115.
  • the binding protein comprises two protein domains: protein domain A and protein domain B.
  • the protein functional region A comprises a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 117 and a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 107;
  • the protein functional region B comprises The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 105.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • the first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 147; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 153.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 136;
  • second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 183.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 147; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 184.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 155;
  • second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 158.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 155;
  • second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 156.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 159;
  • second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 160.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 141; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 142.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 141; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 143.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 141; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 144.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 141; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 145.
  • the binding protein comprises two polypeptide chains: a first polypeptide chain and a second polypeptide chain.
  • first polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 141; the second polypeptide chain includes the amino acid sequence shown in SEQ ID NO: 149.
  • the CDRs mentioned can all include the situation of mutating on the basis of the limited sequence.
  • Said mutation is an insertion, deletion or substitution of 3, 2 or 1 amino acid respectively on the basis of the amino acid sequence of said VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3.
  • amino acid mutation in “insertion, deletion or substitution with 3, 2 or 1 amino acid” refers to the amino acid mutation in the sequence of the variant compared to the original amino acid sequence, including the original amino acid sequence. Amino acid insertions, deletions or substitutions occur on the basis of the sequence.
  • the mutation of CDRs can include mutations of 3, 2 or 1 amino acid, and the same or different numbers of amino acid residues can be optionally selected for mutation among these CDRs, for example, 1 mutation of CDR1 can be performed. 1 amino acid mutation, no amino acid mutation for CDR2 and CDR3.
  • the VH, VL or the polypeptide chain can all include the situation where mutations are made on the basis of the defined sequence.
  • the mutation is a deletion, substitution or addition of one or more amino acid residues in the defined amino acid sequence, and the mutated amino acid sequence has at least 85% sequence identity with the defined amino acid sequence and maintains or The binding activity of the antibody or antigen-binding fragment thereof and binding protein is improved; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most preferably at least 99% sequence identity.
  • the second aspect of the present invention provides an isolated nucleic acid encoding the binding protein according to the first aspect of the present invention.
  • the third aspect of the present invention provides a recombinant expression vector, which comprises the isolated nucleic acid according to the second aspect of the present invention.
  • the expression vector comprises a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
  • the fourth aspect of the present invention provides a transformant comprising the isolated nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention.
  • the host cells of the transformants are prokaryotic cells and/or eukaryotic cells
  • the prokaryotic cells are preferably E. coli cells such as TG1 and BL21
  • the eukaryotic cells are preferably HEK293 cells or CHO cells.
  • the fifth aspect of the present invention provides a method for preparing a binding protein, which comprises culturing the transformant according to the fourth aspect of the present invention, and obtaining the binding protein from the culture.
  • the sixth aspect of the present invention provides a pharmaceutical composition
  • the pharmaceutical composition comprises the binding protein according to the first aspect of the present invention, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes other anti-tumor antibodies as active ingredients.
  • the seventh aspect of the present invention provides a kit comprising the binding protein according to the first aspect of the present invention and/or the pharmaceutical composition according to the sixth aspect of the present invention.
  • the kit further comprises (i) a device for administering the binding protein or pharmaceutical composition; and/or (ii) instructions for use.
  • the eighth aspect of the present invention provides a medicine kit, the medicine kit includes a medicine box 1 and a medicine box 2, and the medicine box 1 includes the binding protein according to the first aspect of the present invention. And/or the pharmaceutical composition according to the sixth aspect of the present invention, the second kit includes other antibodies or pharmaceutical compositions.
  • the ninth aspect of the present invention provides a drug delivery device comprising the binding protein according to the first aspect of the present invention and/or the drug combination according to the sixth aspect of the present invention thing.
  • the drug delivery device further comprises means for containing or administering the synthetin and/or the pharmaceutical composition to a subject, such as a syringe, an infusion set or an implantable drug delivery device.
  • the tenth aspect of the present invention provides a binding protein according to the first aspect of the present invention, a pharmaceutical composition according to the sixth aspect of the present invention, and a reagent according to the seventh aspect of the present invention
  • described cancer is selected from breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, One or more of bladder cancer, sarcoma, colorectal cancer, lymphoma, and multiple myeloma.
  • the eleventh aspect of the present invention provides a method for detecting a specific antigen in vitro or in vivo, which comprises using the binding protein as described in the first aspect of the present invention and/or as described in the sixth aspect of the present invention The described pharmaceutical composition was tested.
  • the twelfth aspect of the present invention provides the binding protein according to the first aspect of the present invention, the pharmaceutical composition according to the sixth aspect of the present invention, and the kit according to the seventh aspect of the present invention , the use of the kit according to the eighth aspect of the present invention, and/or the drug delivery device according to the ninth aspect of the present invention, in diagnosing, preventing and/or treating cancer or other diseases.
  • described cancer is selected from breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, One or more of bladder cancer, sarcoma, colorectal cancer, lymphoma, and multiple myeloma.
  • the thirteenth aspect of the present invention provides a method for diagnosing, preventing and/or treating cancer or other diseases, the method comprising administering the combination as described in the first aspect of the present invention to a patient in need protein, the pharmaceutical composition according to the sixth aspect of the present invention, the kit according to the seventh aspect of the present invention, the kit according to the eighth aspect of the present invention, and/or the ninth aspect of the present invention the steps of the drug delivery device described;
  • described cancer is selected from breast cancer, ovarian cancer, endometrial cancer, kidney cancer, melanoma, lung cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, One or more of bladder cancer, sarcoma, colorectal cancer, lymphoma, and multiple myeloma.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the present invention provides a bispecific binding protein with a Fab-HCAb structure constructed by using the heavy chain antibody (HCAb) and the antigen-binding region Fab of a conventional antibody.
  • the bispecific binding protein molecule of the Fab-HCAb structure in the present invention has a simple and universal structure, and can be applied to a variety of different target combinations; it has smaller molecular weight, fewer polypeptide chains, simple structure, etc. It also has Fc effector functions similar to IgG antibodies, excellent molecular stability and pharmaceutical properties. Moreover, it has advantages over existing bispecific binding proteins with other structures.
  • the molecule of Fab-HCAb structure has one or more of the following advantages compared to the molecule of FIT-Ig structure, VH-IgG structure or IgG-VH structure:
  • the distance between the first binding domain (Fab) and the second binding domain (VH) of the Fab-HCAb structure is more conducive to the interaction between target cells (eg, tumor cells) and effector cells (eg, T cells). Interactions form immune synapses to further promote the activation of effector cells;
  • Fab-HCAb is more compact, and the distance between its two second binding domains (VH) is closer, which is more conducive to the clustering and multimerization of the target in some cases;
  • the Fab-HCAb structure may preferentially bind to the target recognized by the Fab domain, and then cause the binding of the VH domain.
  • the sequence of binding of different targets and the difference in binding force can be applied to the needs of some special application scenarios Fab-HCAbs such as TAA ⁇ 4-1BB can preferentially bind to tumor targets.
  • Figure 2 shows the activity of PD-L1 ⁇ 4-1BB molecule binding to human 4-1BB cells CHO-K1/hu 4-1BB.
  • Figure 3 shows the activity of PD-L1 ⁇ 4-1BB molecule binding to human PD-L1 cells CHO-K1/hPD-L1.
  • FIG. 4 shows that PD-L1 ⁇ 4-1BB molecules activate T cells in a mixed lymphocyte reaction (MLR) experiment: (A) IL-2 release level; (B) IFN- ⁇ release level.
  • MLR mixed lymphocyte reaction
  • Figure 5 shows the activity of B7H4 ⁇ 4-1BB molecule binding to human 4-1BB cells CHO-K1/hu 4-1BB.
  • Figure 6 shows the activity of B7H4 ⁇ 4-1BB molecule binding to tumor cell SK-BR-3.
  • Figure 7 shows that B7H4 ⁇ 4-1BB molecules mediate T cell-specific activation by SK-BR-3 cells.
  • Figure 8 shows that PD-L1 ⁇ 4-1BB molecules mediate T cell-specific activation by CHO-K1/hPD-L1 cells.
  • Figure 9 shows the binding activity of B7H4 ⁇ OX40 molecules to human OX40 cells CHO-K1/hu OX40.
  • Figure 10 shows the activity of B7H4 ⁇ OX40 molecule binding to tumor cell SK-BR-3.
  • Figure 11 shows that B7H4xOX40 molecules mediate T cell specific activation by human B7H4 cells CHO-K1/hB7H4.
  • Figure 12 shows internalization of BCMA binding proteins on NCI-H929 cells.
  • Figure 13 shows the BLI method to determine the affinity of BCMA binding proteins to BCMA: (A) heavy chain antibody PR004433; (B) Fab-HCAb structured bispecific binding protein PR005744.
  • Figure 14 shows the activity of HER2xCTLA4 molecule binding to tumor cell SK-BR-3.
  • Figure 15 shows the activity of HER2xCTLA4 molecule binding to human CTLA4 cells CHO-K1/hCTLA4.
  • Figure 16 shows the pharmacokinetics of the molecule PR004270 of the Fab-HCAb structure in mice.
  • Figure 17 shows the predicted Fab-HCAb structure: (A) three-dimensional structural model of Fab-HCAb, A1 and A2 are the antigen binding sites at the Fab end, B1 and B2 are the antigen binding sites at the VH end; (B) Fab- The relative distance between different antigen binding sites when the HCAb structure is in the most stretched state; (C) the relative distance between different antigen binding sites when the FIT-Ig structure is in the most stretched state.
  • binding protein or "antigen-binding protein” generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen part.
  • An antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both may typically be included.
  • VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4 .
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the three CDRs of VH are denoted as HCDR1, HCDR2 and HCDR3 respectively, and can also be denoted as VH CDR1, VH CDR2 and VH CDR3;
  • the three CDRs of VL are denoted as LCDR1, LCDR2 and LCDR3, respectively, and can also be denoted as VL CDR1, VL CDR2 and VL CDR3.
  • antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugation antibodies, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen binding fragments
  • Fv fragments F(ab') 2
  • scFv di-scFv and/or dAb
  • immunoconjugation antibodies eg, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • the amino acid sequences of the CDRs are shown in accordance with the Chothia definition rules.
  • the CDRs of antibodies can be defined by a variety of methods in the art, such as Kabat's rules of definition based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • the combined definition rule including the Kabat definition and the Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the Combined definition rule is to combine the range defined by Kabat and Chothia, and based on this, a larger range is taken, as shown in the following table. It will be understood by those of skill in the art that, unless otherwise specified, the terms "CDRs" and "complementarity determining regions" of a given antibody or regions thereof (eg, variable regions) are to be understood to encompass the above-mentioned already described above as described by the present invention. complementarity-determining regions defined by any of the known schemes. Although the scope of protection claimed in the present invention is based on the sequence shown in the Chothia definition rule, the amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rule) to bb position (Chothia coding rule);
  • Haa-Hbb can refer to starting from the N-terminus of the antibody heavy chain , the amino acid sequence from position aa (Chothia coding rule) to bb position (Chothia coding rule).
  • L24-L34 may refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain according to the Chothia coding rules Amino acid sequence from position 26 to position 32. It should be known to those skilled in the art that when coding CDRs with Chothia, there may be insertion sites at some positions (see http://bioinf.org.uk/abs/).
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible minor natural mutations.
  • Monoclonal antibodies are usually highly specific for a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • monoclonal denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • monoclonal antibodies used in accordance with the present invention can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • the term "fully human antibody” generally refers to an antibody that is expressed by transferring all or part of a human antibody-encoding gene into a genetically engineered antibody gene-deficient animal. All parts of an antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, and the like.
  • the term “specifically binds” generally refers to the binding of an antibody to an epitope through its antigen binding domain, and that binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody is said to "specifically bind” to an antigen when it is more likely to bind to an epitope through its antigen-binding domain than to a random, unrelated epitope than to a random, unrelated epitope.
  • Epipe refers to a specific group of atoms (eg, sugar side chains, phosphoryl groups, sulfonyl groups) or amino acids on an antigen to which an antigen binding protein (eg, an antibody) binds.
  • Fab generally refers to the antigen-binding portion of a conventional antibody (eg, IgG), including the heavy chain variable region VH, light chain variable region VL and heavy chain constant region domain CH1 and light chain variable region of the antibody.
  • Chain constant region CL In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region of the heavy chain and other constant The domains are linked to form the heavy chain.
  • Fab also refers to variant structures of Fab.
  • the C-terminus of VH is linked to the N-terminus of CL to form a polypeptide chain
  • the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH/VL) structure
  • the CH1 of the Fab is not linked to the hinge region, but the C-terminus of the CL is linked to the hinge region of the heavy chain to form a Fab (cross Fd/LC) structure.
  • VH generally refers to the heavy chain variable region VH domain of an antibody, that is, it can be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals, or it can be Camelidae, etc.
  • the heavy chain variable region VHH of an animal heavy chain antibody (HCAb structure) can also be the heavy chain variable region VH of a fully human heavy chain antibody (HCAb structure) produced by using Harbour HCAb transgenic mice.
  • antigen-binding fragment generally refers to any protein functional region that can specifically bind to an antigen, either "Fab” or "VH”, or other antigen-binding forms (such as liposomes) protein (lipocalins), neural cell adhesion molecule (NCAM), fibronectin (fibronectin), ankyrin repeat fragment protein (DARPins) and other derivative protein structures).
  • Fab fragment-binding forms
  • NCAM neural cell adhesion molecule
  • fibronectin fibronectin
  • DARPins ankyrin repeat fragment protein
  • Fab-HCAb structure refers to the structures shown in Table 1 and Figure 1 as structures (1) and (2).
  • the structure contains two polypeptide chains: polypeptide chain 1, also called short chain, from amino terminus to carboxy terminus, which contains VH_A-CH1; polypeptide chain 2, also called long chain, from amino terminus to carboxy terminus, which contains VL_A- CL-L1-VH_B-L2-CH2-CH3.
  • the structure may also comprise two polypeptide chains: polypeptide chain 1, also called short chain, from amino terminus to carboxyl terminus, which contains VL_A-CL; polypeptide chain 2, also called long chain, from amino terminus to carboxyl terminus, It contains VH_A-CH1-L1-VH_B-L2-CH2-CH3.
  • VH_A and VL_A are the heavy chain variable region and light chain variable region of conventional antibody A, respectively
  • VH_B is the heavy chain variable region of heavy chain antibody B
  • CL is the light chain constant region domain
  • CH1, CH2 and CH3 are the first, second and third domains of the heavy chain constant region, respectively
  • L1 and L2 are linking peptides.
  • L1 may be zero.
  • L2 may be the hinge region of IgG or a linker peptide sequence derived from the hinge region, or the sequence listed in Table 2.
  • Fab-HCAb structure refers specifically to the form of structure (1).
  • tumor antigen may be either tumor specific antigen (TSA) or tumor associated antigen (TAA).
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • Tumor-specific antigens refer to antigens that are unique to tumor cells and do not exist on normal cells or tissues. Tumor-associated antigens are not specific to tumor cells, but also exist in normal cells or tissues, but are highly expressed when tumor cells proliferate.
  • target cells refers to cells that need to be eliminated, mainly tumor cells, but also immunosuppressive cells and the like.
  • effector cells generally refers to immune cells involved in the clearance of foreign antigens and in performing effector functions in an immune response. Such as plasma cells, cytotoxic T cells, NK cells and so on.
  • PD-L1 generally refers to programmed death ligand 1 protein, functional variants thereof and/or functional fragments thereof.
  • PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by (in humans) the CD274 gene.
  • CD274 cluster of differentiation 274
  • B7-H1 B7 homolog 1
  • PD-L1 sequences are known in the art.
  • the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7; an exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found under NCBI Accession No.
  • PD-L1 is mainly expressed in antigen presenting cells and various tumor cells.
  • the interaction between PD-L1 and PD-1 can down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive effect.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expressed PD-L1 is beneficial to the occurrence and growth of tumors and induces anti-tumor effects. apoptosis of T cells and further protect tumor cells from immune attack.
  • HER2 generally refers to the receptor tyrosine kinase erbB-2 (also known as ERBB2), functional variants thereof and/or functional fragments thereof.
  • HER2 sequences are known in the art. For example, an exemplary full-length human HER2 sequence can be found in Uniprot accession number P04626; an exemplary full-length cynomolgus monkey HER2 sequence can be found in NCBI accession number XP_005584091.
  • B7H4 generally refers to V-Set domain-containing T cell activation inhibitory factor 1 (also known as VTCN1, B7h.5, B7S1, B7x), functional variants and/or functional fragments thereof.
  • B7H4 sequences are known in the art. For example, an exemplary full-length human B7H4 sequence can be found in Uniprot Accession No. Q7Z7D3; an exemplary full-length cynomolgus monkey B7H4 sequence can be found in NCBI Accession No. XP_005542249; an exemplary full-length mouse B7H4 sequence It can be found under Uniprot accession number Q7TSP5.
  • B7-H4 is a transmembrane protein belonging to the B7/CD28 superfamily.
  • B7-H4 protein is expressed in some immune cells such as monocytes and dendritic cells, and may be involved in the negative regulation of immune responses of T cells.
  • B7H4 is also highly expressed on the surface of tumor cells of breast cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, renal cancer, etc., while it is not expressed or expressed very low in most normal tissues.
  • B7-H4 has received attention in recent years.
  • Anti-B7-H4 antibodies can be applied to tumor cells through various mechanisms, but their research and development direction is mainly focused on monoclonal antibodies, and there is currently no bispecific antibody therapy.
  • 4-1BB generally refers to tumor necrosis factor receptor superfamily member 9 (also known as CD137, TNFRSF9, 4-1BBL receptor), functional variants and/or functional fragments thereof.
  • the 4-1BB sequence is known in the art.
  • an exemplary full-length human 4-1BB sequence can be found in Uniprot Accession No. Q07011; an exemplary full-length cynomolgus monkey 4-1BB sequence can be found in NCBI Accession No. XP_005544945.
  • 4-1BB is a transmembrane protein belonging to the TNF receptor superfamily.
  • 4-1BB is a costimulatory molecule expressed on a variety of immune cells and is a multifunctional regulator of immune activity.
  • 4-1BB activates T cells through trimerization mediated by its ligand 4-1BBL, promoting cell proliferation and cytokine release.
  • Agonistic antibodies against 4-1BB have the function of inhibiting tumors.
  • the earliest 4-1BB antibodies entered clinical trials are Pfizer's Utomilumab and Bristol-Myers Squibb's (BMS) Urelumab (BMS-663513).
  • BMS Bristol-Myers Squibb's
  • the initial clinical results of Urelumab were published in 2008, and although encouraging efficacy was observed in some patients, data showed that Urelumab caused liver toxicity that was target- and dose-related.
  • Utomilumab is safer, and the dose can be increased to 10 mg/kg, but the therapeutic effect is still poor.
  • the core issue of 4-1BB targeted drug development is how to properly activate immune cells through 4-1BB to achieve a balance between efficacy and safety.
  • OX40 generally refers to tumor necrosis factor receptor superfamily member 4 (also known as CD134, TNFRSF4, OX40L receptor), functional variants and/or functional fragments thereof.
  • OX40 sequences are known in the art. For example, an exemplary full-length human OX40 sequence can be found in Uniprot accession number P43489; an exemplary full-length cynomolgus monkey OX40 sequence can be found in NCBI accession number XP_005545179.
  • OX40 a member of the TNF receptor superfamily, is involved in enhancing T cell responses triggered by T cell receptors and is a co-stimulatory receptor molecule. It is a 50kD transmembrane protein.
  • OX40 is transiently expressed on human CD4 + and CD8 + T cells after TCR stimulation. However, at the tumor site, OX40 expressed on CD4 + T cells than CD8 + T cells were higher. Therefore, CD4 + and CD8 + T cells are potential targets for OX40-directed immunotherapy drugs for cancer.
  • Antibodies to OX40 Several preclinical studies have shown that mAbs against OX40 produce deleterious immunosuppressive side effects by promoting the accumulation of MDSCs and the production of Th2 cytokines.
  • BCMA generally refers to tumor necrosis factor receptor superfamily member 17 (also known as B-cell maturation antigen, TNFRSF17, CD269), functional variants and/or functional fragments thereof.
  • BCMA sequences are known in the art. For example, an exemplary full-length human BCMA sequence can be found in Uniprot Accession No. Q02223; an exemplary full-length cynomolgus monkey BCMA sequence can be found in NCBI Accession No. XP_005591343.
  • BCMA is a transmembrane protein belonging to the TNF receptor superfamily that is involved in B cell maturation, growth and survival.
  • BCMA is expressed in the malignant plasma cells of patients with multiple myeloma (MM) and supports the growth and survival of multiple myeloma cells. Multiple myeloma is the second most common hematological malignancy after non-Hodgkin's lymphoma, accounting for about 13% of hematological malignancies.
  • APRIL high-affinity ligand APRIL
  • BAFF low-affinity ligand BAFF.
  • BCMA is expressed in the malignant plasma cells of patients with multiple myeloma (MM) and supports the growth and survival of multiple myeloma cells. Multiple myeloma is the second most common hematological malignancy after non-Hodgkin's lymphoma, accounting for about 13% of hematological malignancies.
  • BCMA antibodies can target MM cells through a variety of mechanisms.
  • CTLA4 generally refers to cytotoxic T lymphocyte-associated protein-4 (also known as CD152), functional variants thereof and/or functional fragments thereof.
  • CTLA4 sequences are known in the art. For example, an exemplary full-length human CTLA4 sequence can be found in Uniprot Accession No. P16410; an exemplary full-length cynomolgus CTLA4 sequence can be found in Uniprot Accession No. G7PL88.
  • CTLA4 is a negative regulator expressed on T cells. After it binds to CD80 or CD86 on antigen-presenting cells, it blocks the co-stimulatory signal of CD28 and at the same time downregulates the activity of T cells and plays an immunosuppressive role.
  • Ipilimumab monoclonal antibody (trade name ) is the first approved anti-CTLA4 monoclonal antibody. Ipilimumab has shown a good therapeutic effect in the treatment of advanced melanoma, but Ipilimumab also brings high immune-related side effects, which seriously affects its clinical application. Most of the toxic and side effects exhibited by Ipilimumab are related to the CTLA4 target. In the current combination regimen of PD-1/PD-L1 inhibitors and CTLA4 inhibitors, CTLA4 inhibitors, whether Ipilimumab or Tremelimumab, are usually selected. low dose.
  • CTLA4 inhibitors In order to reduce the toxic and side effects of CTLA4 inhibitors, one of the methods worth trying is to deliver CTLA4 inhibitors into tumor tissues, so that the relevant T cell-mediated responses are limited to the tumor microenvironment and reduce cytokine release. Syndrome risk.
  • the use of antibodies that recognize tumor-associated antigens can redirect CTLA4 inhibitors to specific tumor microenvironments, so that they can relieve T cells from immunosuppressive signals in the tumor microenvironment and restore T cell function.
  • the present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting protein-encoding genes into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are useful for this study. It is well known to those of ordinary skill in the art and described in numerous publications.
  • the experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.
  • Table 1 and FIG. 1 in this example list the structures of the bispecific binding proteins constructed by the heavy chain antibody (HCAb) and its derived single domain antibody (sdAb) involved in the application of the present invention. Each structure is described further below.
  • the Fab-HCAb structure is the structure shown in Table 1 and Figure 1 as structure (1) and structure (2), and the preferred structure is structure (1).
  • the domains are linked by linker peptides.
  • amino acid mutations are introduced in the Fc region of the heavy chain to alter its binding to Fc receptors and thereby alter the associated effector function or other properties.
  • Table 2 lists the linker peptide sequences that may be used in the structural design of this application.
  • the present invention provides a method for constructing bispecific binding proteins using two parental monoclonal antibodies: conventional antibody A (eg, IgG antibody) that binds to a first antigen and heavy chain antibody B that binds a second antigen.
  • conventional antibody A eg, IgG antibody
  • heavy chain antibody B binds a second antigen
  • the Fab end is derived from conventional antibody A
  • VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of antibody A, respectively.
  • the VH end is derived from heavy chain antibody B
  • VH_B is the heavy chain variable region of heavy chain antibody B.
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of the heavy chain constant region.
  • h is the hinge region or derived sequence of an IgG antibody
  • L or L1 or L2 is the linking peptide.
  • the binding protein of structure (1) contains two distinct polypeptide chains: polypeptide chain 1, also called short chain, from the amino terminus to carboxy terminus, which contains VH_A-CH1; polypeptide chain 2, also called long chain, from the amino terminus to the carboxy terminus. Carboxyl terminus, which contains VL_A-CL-L1-VH_B-L2-CH2-CH3.
  • polypeptide chain 1 also called short chain, from the amino terminus to carboxy terminus, which contains VH_A-CH1
  • polypeptide chain 2 also called long chain, from the amino terminus to the carboxy terminus.
  • Carboxyl terminus which contains VL_A-CL-L1-VH_B-L2-CH2-CH3.
  • the VL_A of antibody A and the VH_B of heavy chain antibody B are fused on the same polypeptide chain, which can avoid mismatched by-products generated by the association of VL_A and VH_B.
  • VH_B of polypeptide chain 2 is linked to CH2 via linker peptide L2;
  • L2 can be the hinge region of IgG or a linker peptide sequence derived from the hinge region, or a sequence listed in Table 2, preferably a human IgG1 hinge or a human IgG1 hinge (C220S) or Sequence of G5-LH.
  • the CL of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • the CL of polypeptide chain 2 is linked to VH_B via a linking peptide L1; L1 may be the sequence listed in Table 2.
  • the binding protein of structure (2) contains two distinct polypeptide chains: polypeptide chain 1, also called short chain, from amino terminus to carboxyl terminus, which contains VL_A-CL; polypeptide chain 2, also called long chain, from amino terminus to carboxyl terminus. Carboxyl terminus, which contains VH_A-CH1-L1-VH_B-L2-CH2-CH3.
  • VH_B of polypeptide chain 2 is linked to CH2 via linker peptide L2;
  • L2 can be the hinge region of IgG or a linker peptide sequence derived from the hinge region, or a sequence listed in Table 2, preferably a human IgG1 hinge or a human IgG1 hinge (C220S) or Sequence of G5-LH.
  • CH1 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L1 is 0.
  • CH1 of polypeptide chain 2 is linked to VH_B via a linker peptide L1; L1 may be the sequence listed in Table 2.
  • the binding protein of structure (3) contains two distinct polypeptide chains: polypeptide chain 1, also called short chain, from amino terminus to carboxyl terminus, which contains VL_A-CL; polypeptide chain 2, also called long chain, from amino terminus to carboxyl terminus. Carboxyl terminus, which contains VH_A-CH1-h-CH2-CH3-L-VH_B.
  • CH3 of polypeptide chain 2 is directly fused to VH_B, that is, the length of L is 0.
  • CH3 of polypeptide chain 2 is linked to VH_B via a linker peptide L; L may be the sequence listed in Table 2.
  • the binding protein of structure (4) contains two polypeptide chains: polypeptide chain 1, also called short chain, from amino terminus to carboxyl terminus, which contains VL_A-CL; polypeptide chain 2, also called long chain, from amino terminus to carboxyl terminus , which contains VH_B-L-VH_A-CH1-h-CH2-CH3.
  • VH_B of polypeptide chain 2 is directly fused to VH_A, that is, the length of L is 0.
  • VH_B of polypeptide chain 2 is linked to VH_A via a linker peptide L; L may be the sequence listed in Table 2.
  • the diabody molecule of FIT-Ig structure can be constructed from: conventional antibody A that binds to the first antigen and conventional antibody B that binds to the second antigen.
  • the binding protein of structure (5) contains three polypeptide chains: polypeptide chain 1, from amino terminus to carboxyl terminus, which contains VL_A-CL-L-VH_B-CH1-h-CH2-CH3; polypeptide chain 2, from amino terminus to carboxyl terminus terminus, which contains VH_A-CH1; polypeptide chain 3, from amino terminus to carboxy terminus, which contains VL_B-CL.
  • VH_A and VL_A are the heavy chain variable region and light chain variable region of antibody A, respectively
  • VH_B and VL_B are the heavy chain variable region and light chain variable region of antibody B, respectively
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains of the heavy chain constant region, respectively, h is the hinge region or derived sequence of an IgG antibody, and L is the linking peptide.
  • h is the hinge region or derived sequence of an IgG antibody, and L is the linking peptide.
  • L is the linking peptide.
  • CL of polypeptide chain 1 is directly fused to VH_B, that is, the length of L is 0.
  • CL of polypeptide chain 1 is linked to VH_B via a linker peptide L; L may be the sequence listed in Table 2.
  • This example describes a general method for antibody preparation using mammalian host cells (eg, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives), transient transfection expression, and affinity capture isolation.
  • mammalian host cells eg, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derivatives
  • transient transfection expression e.g., transient transfection expression
  • affinity capture isolation e.g., affinity capture isolation.
  • This method is suitable for target antibody containing Fc; the target antibody can be composed of one or more protein polypeptide chains; it can be derived from one or more expression plasmids.
  • the amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence by a codon optimization method; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with host cells.
  • the plasmid encoding the antibody polypeptide chain is simultaneously transfected into mammalian host cells according to a specific ratio, and the recombinant antibody with correct folding and polypeptide chain assembly can be obtained by using conventional recombinant protein expression and purification techniques. Specifically, FreeStyle TM 293-F cells (Thermo, #R79007) were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504).
  • the cell concentration was adjusted to 6-8x10 5 cells/ml and incubated for 24 hours at 37°C in an 8% CO 2 shaker at a cell concentration of 1.2x10 6 cells/ml.
  • Opti-MEM 1.5 ml of Opti-MEM was dissolved in 120 ⁇ l of 1 mg/ml PEI (Polysciences, #23966-2), and it was left to stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop the plasmid PEI mixed solution while shaking the culture flask, and culture at 37°C in an 8% CO 2 shaker for 5 days. Cell viability was observed after 5 days. The culture was collected, centrifuged at 3300g for 10 minutes, and the supernatant was taken; then the supernatant was centrifuged at high speed to remove impurities.
  • PEI Polysciences, #23966-2
  • This example uses analytical size exclusion chromatography (SEC) to analyze protein samples for purity and aggregate form.
  • An analytical column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8 mm x 30 em) was attached to a high pressure liquid chromatograph HPLC (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer for at least 1 hour at room temperature.
  • HPLC Analogent Technologies, Agilent 1260 Infinity II
  • An appropriate amount of protein sample (at least 10 ⁇ g) was filtered through a 0.22 ⁇ m filter and injected into the system, and the HPLC program was set: the sample was flowed through the column with PBS buffer at a flow rate of 1.0 ml/min for a maximum time of 25 minutes.
  • the HPLC will generate an analytical report reporting the retention times of components of different molecular sizes within the sample.
  • IgG monoclonal antibody and HCAb monoclonal antibody The information of IgG monoclonal antibody and HCAb monoclonal antibody is listed in Table 3, its sequence number is shown in Table 6, and its amino acid sequence is shown in Table 11.
  • a bispecific binding protein with a Fab-HCAb structure was designed according to the structures described in Example 1.1.1 and Figure 1(1) or the structures described in Example 1.1.2 and Figure 1(2), and its molecular design is summarized in Table 4 , its sequence number is shown in Table 7, and its amino acid sequence is shown in Table 12; and the protein samples were prepared and analyzed according to the method described in Example 2, which are summarized in Table 9.
  • Table 8 also lists the sequence numbers of the corresponding CDR sequences of protein domain A (the first antigen binding domain) and protein domain B (the second antigen binding domain) of the bispecific binding protein.
  • amino acid mutations are introduced in the Fc region of the heavy chain to alter its binding to Fc receptors and thereby alter the associated effector function or other properties.
  • the mutation site codes in the table are: AAG: (L234A, L235A, P329G); LALA: (L234A, L235A).
  • PD-L1 ⁇ 4-1BB can activate T cells by blocking the PD-1/PD-L1 signaling pathway.
  • PD-L1 molecules highly expressed on the surface of tumor cells can use PD-L1 ⁇ 4-1BB to promote the cross-linking and trimerization of 4-1BB molecules on the surface of T cells and activate downstream signaling pathways, thereby promoting T cells activation and proliferation.
  • PD-L1 ⁇ 4-1BB-mediated T cell activation is limited to the tumor microenvironment, which can avoid the toxic side effects caused by over-activation of T cells in normal tissues by monoclonal antibodies like Urelumab.
  • Example 4.1.1 Obtaining fully human IgG antibody against PD-L1
  • the candidate antibody molecules are then subjected to sequence analysis and optimization, resulting in several variant sequences.
  • the VL and VH sequences of the antibody are fused and expressed with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules.
  • the human VH gene was amplified from plasma cells by conventional molecular biology methods, and the amplified human VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the heavy chain Fc sequence of human IgG1 antibody.
  • the plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the supernatant of fully human HCAb antibody is obtained.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the binding of HCAb antibody supernatant to CHO-K1 cell CHO-K1/hu4-1BB highly expressing human 4-1BB was tested by FACS, and positive HCAb antibody was identified.
  • These HCAb antibodies were further identified, and several candidate HCAb antibody molecules were selected according to their binding ability to human 4-1BB, cynomolgus monkey 4-1BB binding ability, T cell activation ability and other parameters.
  • this example utilizes the Fab of the anti-PD-L1 IgG antibody PR000265 and the VH of the anti-4-1BB HCAb antibody PR001760 to construct a Fab-HCAb structure as described in Example 1.1.1 ( Figure 1 Structure ( 1): bispecific binding proteins PR004270 and PR007164 against PD-L1 ⁇ 4-1BB of Fab(CL)-VH-Fc); (2): Anti-PD-L1 ⁇ 4-1BB bispecific binding protein PR007163 of Fab(CH1)-VH-Fc).
  • PR004270 The molecular designs of PR004270, PR007163 and PR007164 are shown in Table 4, and the corresponding sequence numbers are shown in Table 7; the molecules were prepared and analyzed according to the method described in Example 2, and are summarized in Table 9. As shown in Table 9, the purified yields of PR007164 (structure (1)), PR004270 were significantly higher than those of PR007163 (structure (2)).
  • this example also utilizes the Fab of the anti-PD-L1 IgG antibody PR000265 and the VH of the anti-4-1BB HCAb antibody PR001760 to construct an anti-PD with an IgG-VH structure as described in Example 1.
  • -Bispecific binding protein PR003550 for L1 ⁇ 4-1BB The molecular design of PR003550 is shown in Table 5, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 10.
  • this example also utilizes the Fab of the anti-PD-L1 IgG antibody PR000265 and the VH of the anti-4-1BB HCAb antibody PR001760 to construct an anti-PD with a VH-IgG structure as described in Example 1.1.4 -
  • the bispecific binding protein PR004268 for L1 ⁇ 4-1BB The molecular design of PR004268 is shown in Table 5, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 10.
  • this example also utilizes the Fab of the anti-PD-L1 IgG antibody PR000265 and the Fab of the anti-4-1BB IgG antibody PR000197 to construct the anti-PD with the FIT-Ig structure described in Example 1.2.1 -
  • the bispecific binding protein PR000701 of L1 ⁇ 4-1BB is shown in Table 5, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 10.
  • flow cytometry FACS was used to test the binding ability of the binding protein to CHO-K1 cell line CHO-K1/hu4-1BB (Nanjing GenScript, M00538) cells highly expressing human 4-1BB. Specifically, digestion of the cells and resuspended with complete medium; cell density was adjusted to 2x10 6 cells / mL. The cells were then seeded in a 96-well V-bottom plate (Corning, #3894) at 100 ⁇ L/well (2 ⁇ 10 5 cells/well), centrifuged at 4° C. for 5 minutes, and the supernatant was discarded. Then, 100 ⁇ L/well of the serially diluted binding protein was added to the 96-well plate and mixed well.
  • the binding protein can be diluted from the highest final concentration of 200 nM to a total of 12 concentrations in a 3-fold concentration gradient; hIgG1 iso (CrownBio, #C0001) was used as the isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. Then, 100 ⁇ L/well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA) was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the binding protein to the target cells and the EC50 value were obtained by four-parameter nonlinear fitting.
  • the positive control molecule is anti-4-1BB monoclonal antibody Urelumab (protein number PR000628) or anti-4-1BB HCAb antibody PR001760.
  • the PD-L1 x 4-1BB bispecific binding proteins (PR004270, PR004268, PR003550) were comparable in their ability to bind 4-1BB and were superior to the positive control Urelumab in MFI maxima and in EC50 values Molecular PR000701 which is superior to FIT-Ig structure.
  • the PD-L1 ⁇ 4-1BB bispecific binding proteins (PR007163, PR007164) were comparable in their ability to bind 4-1BB to their parental mAb PR001760.
  • flow cytometry FACS was used to test the binding ability of the binding protein to the CHO-K1 cell line CHO-K1/hPD-L1 (Nanjing GenScript, M00543) that highly expresses human PD-L1. Specifically, digestion of the cells and resuspended with complete medium; cell density was adjusted to 1x10 6 cells / mL. Next, the cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 ⁇ L/well, centrifuged at 4° C. for 5 minutes, and the supernatant was discarded. Then, 100 ⁇ L/well of the serially diluted binding protein was added to the 96-well plate and mixed well.
  • the binding protein can be diluted from the highest final concentration of 200 nM to a total of 12 concentrations in a 3-fold concentration gradient; hIgG1 iso (CrownBio, #C0001) was used as the isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. Then, 100 ⁇ L/well of pre-chilled FACS buffer (PBS buffer containing 0.5% BSA) was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • Fluorescence signal values were read using a BD FACS CANTOII flow cytometer or an ACEA NovoCyte flow cytometer, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the binding protein to the target cells and the EC50 value were obtained by four-parameter nonlinear fitting.
  • the positive control molecule is the anti-PD-L1 monoclonal antibody PR000265, which is also the parental monoclonal antibody at the PD-L1 end of PD-L1 ⁇ 4-1BB.
  • the ability of the Fab-HCAb structure molecule (PR004270) and the VH-IgG structure molecule (PR004268) to bind to PD-L1 is similar to that of the parent mAb PR000265, although the EC50 value of its binding to PD-L1 is slightly weaker than that of the parent monoclonal antibody PR000265.
  • the parental mAb but with a higher MFI maximum for binding than the parental mAb.
  • the IgG-VH-structured molecule (PR003550) binds PD-L1 similarly to the parent mAb PR000265, and the EC50 value and MFI maximum are slightly better than those of the FIT-Ig-structured molecule (PR000701).
  • the Fab-HCAb-structured molecules (PR007163, PR007164) were comparable in their ability to bind PD-L1 as the parental mAb PR000265.
  • mixed lymphocyte reaction was used to study the activation effect of PD-L1 ⁇ 4-1BB bispecific binding protein on T cells.
  • CD14 magnetic beads (Meltenyi, #130-050-201) were used to separate monocytes from the first donor PBMC cells (Miaotong Biotechnology); for specific operations, refer to the relevant kit instructions. Then, 50 ng/mL recombinant human IL-4 (PeproTech, #200-02-A) and 100 ng/mL recombinant human GM-CSF (PeproTech, #300-03-A) were added and induced at 37°C for 7 days to obtain Immature dendritic cells (iDC cells).
  • iDC cells Immature dendritic cells
  • mDC cells mature dendritic cells
  • LPS lipopolysaccharide Lipopolysaccharide
  • mDC cells mature dendritic cells
  • T lymphocytes were isolated from the second donor PBMC cells (Miaotong Bio) by using a T cell isolation kit (Meltenyi, #130-096-535).
  • the obtained T cells and mDC cells were seeded into a 96-well plate at a ratio of 5:1 (1 ⁇ 10 5 /well of T cells and 2 ⁇ 10 4 /well of mDC cells).
  • the IFN- ⁇ concentration in the supernatant on day 5 was detected by a ⁇ ELISA kit (Thermo, #88-7316-77).
  • the ELISA detection method refers to the relevant kit operation instructions.
  • the software GraphPad Prism 8 was used for data processing and graph analysis.
  • anti-4-1BB mAb had limited activation of T cells and weak production of cytokines (IFN- ⁇ , IL-2); however, anti-PD- L1 monoclonal antibody (PR000265) has obvious activation effect.
  • PD-L1 ⁇ 4-1BB bispecific binding protein can further improve the function of T cells, which is superior to anti-PD-L1 mAb.
  • the IgG-VH structure molecule PR003550
  • the Fab-HCAb structure molecule PR004270
  • the target cells can be CHO-K1/hPD-L1 cells that highly express human PD-L1 (Nanjing GenScript, M00543); the effector cells can be isolated human PBMCs or T cells.
  • 0.3 ⁇ g/mL anti-CD3 antibody OKT3 (Thermo, #16-0037-81) was coated on a 96-well plate (Corning, #3599) at 100 ⁇ L/well.
  • the density of human T cells isolated from human PBMC using a T cell sorting kit (Miltenyi, #130-096-535) was adjusted to 2 ⁇ 10 6 cells/mL, and the density of target cells was adjusted to 3 ⁇ 10 5 cells/mL, and then the two cell suspensions were seeded in a 96-well plate at 50 ⁇ L/well, and the final effect-target ratio was 20:3.
  • the 96 well plate was placed 37 °C, 5% CO 2 incubator for 3 days.
  • the supernatants after 48 hours and 72 hours of culture were collected respectively, and the IL-2 concentration in the supernatants after 48 hours was detected by IL-2 ELISA kit (Thermo, #88-7025-88), and the IL-2 concentration in the supernatants after 48 hours was detected by IFN- ⁇ ELISA kit. (Thermo, #88-7316-77)
  • the IFN- ⁇ concentration in the supernatant after 72 hours was measured.
  • the ELISA detection method refers to the relevant kit operation instructions. Data processing and graph analysis were performed using the software GraphPad Prism 8.
  • the cross-linking-independent anti-4-1BB mAb Urelumab can activate T cells to release IFN- ⁇ ;
  • Example 4.5 and Example 4.6 the molecule of Fab-HCAb structure (PR004270) showed stronger T cell activation ability than the molecule of FIT-Ig structure (PR000701).
  • the known human IgG1 full-length antibody crystal structure (PDB accession number 1HZH) was used to predict the Fab-HCAb (structural (1)) and the three-dimensional structural model of FIT-Ig (structure (5)), and on this basis, the relative distances between different antigen-binding sites were measured (Fig. 17( B) and (C)); in these two structural models, the linking peptide GS_7 (SEQ ID NO: 163) with a length of 7 amino acids is used for linking between protein functional domain A and protein functional domain B.
  • the Fab-HCAb structure is more compact.
  • the distance between the two VH ends (B1 and B2) is about 10 nm, and the distance between the two Fab ends (A1 and A2) is about 30 nm; correspondingly, in FIT -In the Ig structure, the distance between B1 and B2 is about 18 nm, and the distance between A1 and A2 is about 37 nm.
  • this more compact structural property of Fab-HCAb may be more favorable for 4-1BB to trimerize and cluster on the cell surface, thereby activating downstream signaling.
  • B7H4 ⁇ 4-1BB with a Fab-HCAb, IgG-VH or VH-IgG structure targeting B7H4 and 4-1BB through one or more mechanisms of action Improve anti-tumor efficacy and safety.
  • B7H4 ⁇ 4-1BB can activate T cells by deactivating the negative regulatory signal of B7H4.
  • B7H4 ⁇ 4-1BB is enriched in tumor tissues with high expression of B7H4.
  • B7H4 ⁇ 4-1BB In the tumor microenvironment, immune cells and tumor cells are combined through B7H4 ⁇ 4-1BB to promote the formation of immune synapses; at the same time, high expression B7H4 molecules on the surface of tumor cells can promote the cross-linking of 4-1BB molecules on the surface of T cells through B7H4 ⁇ 4-1BB, and activate downstream signaling pathways to provide costimulatory signals, thereby promoting the activation and proliferation of T cells, and improving the anti-tumor effect. tumor activity.
  • B7H4 ⁇ 4-1BB can only use target cells in the tumor microenvironment to mediate T cell activation, so as to avoid the toxic and side effects caused by the excessive activation of T cells in normal tissues by monoclonal antibodies similar to Urelumab.
  • the anti-4-1BB fully human HCAb antibody PR001760 (Table 6) used in this example was derived from Harbour HCAb mice, and the discovery process was as described in Example 4.1.3.
  • this example utilizes the Fab of the anti-B7H4 IgG antibody PR002408 and the VH of the anti-4-1BB HCAb antibody PR001760 to construct an anti-B7H4 ⁇ 4-1BB having a Fab-HCAb structure as described in Example 1.1.1
  • the bispecific binding protein PR004279 The molecular design of PR004279 is shown in Table 4, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 9.
  • this example also utilizes the Fab of the anti-B7H4 IgG antibody PR002408 and the VH of the anti-4-1BB HCAb antibody PR001760 to construct the anti-B7H4 ⁇ 4 with the IgG-VH structure described in Example 1.1.3 -1BB bispecific binding protein PR003335.
  • the molecular design of PR003335 is shown in Table 5, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 10.
  • this example also utilizes the Fab of the anti-B7H4 IgG antibody PR002408 and the VH of the anti-4-1BB HCAb antibody PR001760 to construct the anti-B7H4 ⁇ 4 with the VH-IgG structure described in Example 1.1.4 -1BB bispecific binding protein PR004278.
  • the molecular design of PR004278 is shown in Table 5, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 10.
  • Example 4.3 the method described in Example 4.3 was used to test the binding ability of the binding protein to CHO-K1 cell line CHO-K1/hu4-1BB (Nanjing GenScript, M00538) cells highly expressing human 4-1BB.
  • B7H4 ⁇ 4-1BB bispecific binding proteins can all bind 4-1BB; and Fab-HCAb-structured molecules (PR004279) and VH-IgG-structured molecules (PR004278)
  • the ability to bind 4-1BB was superior to that of the IgG-VH structured molecule (PR003335).
  • flow cytometry FACS was used to test the binding ability of the binding protein to the tumor cell line SK-BR-3 (ATCC, HTB-30) highly expressing human B7H4. Specifically, digestion of SK-BR-3 cells were resuspended with complete medium, cell density was adjusted to 2x10 6 cells / mL; followed by 50 ⁇ L cells / well in 96-well V bottom plate (Corning, # 3894). Then, 50 ⁇ L/well of 5-fold serially diluted binding protein was added at a total of 8 concentrations, and mixed well; hIgG1 iso (CrownBio, #C0001) was used as an isotype control. The cells were placed at 4°C and incubated in the dark for 2 hours.
  • the cells were then washed twice with 100 ⁇ L/well of pre-chilled PBS buffer, then centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • fluorescent secondary antibody Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific, Jackson ImmunoResearch, #109-605-098, 1:1000 dilution
  • was added at 100 ⁇ L/well and incubated at 4°C in the dark for 1 Hour.
  • the cells were then washed twice with 100 ⁇ L/well of pre-chilled PBS buffer, then centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the binding protein to the target cells and the EC50 value were obtained by four-parameter nonlinear fitting.
  • the B7H4 ⁇ 4-1BB bispecific binding proteins (PR004279, PR004278, PR003335) can all bind B7H4; and the Fab-HCAb structure molecule (PR004279) binds B7H4 slightly better than other structures.
  • the target cells can be cells SK-BR-3 (ATCC, HTB-30) that highly express human B7H4; the effector cells can be isolated human PBMCs or T cells.
  • the anti-CD3 antibody OKT3 (Thermo, #16-0037-81) was firstly coated on a 96-well plate (Corning, #3799). Next, the density of human T cells was adjusted to 3 ⁇ 10 6 cells/mL, and the density of target cells was adjusted to 3 ⁇ 10 5 cells/mL, and then the two cell suspensions were seeded in 96-well plates at 50 ⁇ L/well. The ratio is 10:1. Then, 5-fold concentration-diluted binding protein was added at 50 ⁇ L/well for a total of 5 concentrations, the maximum final concentration was 6 nM, and two replicate wells were loaded; 30 nM hIgG1 iso (CrownBio, #C0001) was used as a control.
  • the 96 well plate was placed 37 °C, 5% CO 2 incubator.
  • the supernatants after 48 hours and 72 hours of culture were collected respectively, and the IL-2 concentration in the supernatants after 48 hours was detected by IL-2 ELISA kit (Thermo, #88-7025-88), and the IL-2 concentration in the supernatants after 48 hours was detected by IFN- ⁇ ELISA reagent.
  • a cassette (Thermo, #88-7316-77) was used to measure the concentration of IFN- ⁇ in the supernatant after 72 hours.
  • the ELISA detection method refers to the relevant kit operation instructions. Data processing and graph analysis were performed using the software GraphPad Prism 8.
  • the positive control molecule is the anti-4-1BB monoclonal antibody Urelumab.
  • FIG. 7 shows that binding proteins activate T cells to release IL-2.
  • Fab-HCAb structure molecule (PR004279) and IgG-VH structure molecule (PR003335) have stronger ability to activate T cells than Urelumab, and PR004279 is slightly stronger than PR003335.
  • the molecule of VH-IgG structure (PR004278) has a strong ability to bind 4-1BB, it hardly activates T cells. This indicates that when the 4-1BB binding domain VH is located at the N-terminus of the IgG heavy chain, the distance between its target cell binding domain Fab and the 4-1BB binding domain VH is not suitable for the interaction between target cells and T cells .
  • the order of T cell activation ability PR004279>PR003335>Urelumab>PR004278.
  • bispecific binding protein B7H4 ⁇ OX40 with a Fab-HCAb or IgG-VH structure targeting B7H4 and OX40 using a similar mechanism of action to B7H4 ⁇ 4-1BB, through tumor-associated antigens B7H4 redirects OX40 antibodies to tumor cells, specifically activating immune responses in the tumor microenvironment.
  • the anti-B7H4 recombinant fully human IgG antibody PR002408 (Table 6) used in this example was derived from Harbour H2L2 mice, and the discovery process was as described in Example 5.1.1.
  • the anti-OX40 fully human HCAb antibody PR002067 (Table 6) used in this example is derived from Harbour HCAb mice, and its discovery process is similar to the discovery process of the anti-4-1BB HCAb described in Example 4.1.3, that is, using HarbourHCAb mice were immunized with recombinant human OX40-Fc fusion protein (provided by wisdom Chemical) or cell line HEK293/OX40 (provided by wisdom Chemical) with high expression of human OX40, and obtained after multiple rounds of screening.
  • this example utilizes the Fab of the anti-B7H4 IgG antibody PR002408 and the VH of the anti-OX40 HCAb antibody PR002067 to construct an anti-B7H4 ⁇ OX40 bispecific having the Fab-HCAb structure described in Example 1.1.1 Binding protein PR004277.
  • the molecular design of PR004277 is shown in Table 4, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 9.
  • this example also utilizes the Fab of the anti-B7H4 IgG antibody PR002408 and the VH of the anti-OX40 HCAb antibody PR002067 to construct an anti-B7H4 ⁇ OX40 bilayer having an IgG-VH structure as described in Example 1.1.3 Specific binding protein PR004276.
  • the molecular design of PR004276 is shown in Table 5, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 10.
  • flow cytometry FACS was used to test the binding ability of the binding protein to CHO-K1 cell line CHO-K1/huOX40 (Nanjing GenScript, M00561) cells highly expressing human OX40. Specifically, cells were digested and resuspended with F12K complete medium, and the cell density was adjusted to 1 ⁇ 10 6 cells/ml, respectively. 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), followed by the addition of 100 ⁇ L/well, 2 times the final concentration of the 3-fold dilution of the binding protein to be tested. The cells were placed at 4°C and incubated in the dark for 1 hour.
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the binding protein to the target cells and the EC50 value were obtained by four-parameter nonlinear fitting.
  • the positive control molecule was anti-OX40 monoclonal antibody Pogalizumab (protein number PR003475).
  • the B7H4 x OX40 bispecific binding proteins (PR004277, PR004276) can both bind OX40 with comparable binding abilities.
  • Example 5.4 the method described in Example 5.4 was used to test the binding ability of the binding protein to the tumor cell line SK-BR-3 (ATCC, HTB-30) that highly expresses human B7H4.
  • the positive control molecule is the anti-B7H4 mAb PR002408, which is also the parent mAb at the B7H4 end of B7H4 ⁇ OX40.
  • the target cells can be CHO-K1/hB7H4 cells with high expression of human B7H4 (manufactured by Hebo Pharmaceuticals); the effector cells can be isolated human PBMCs or T cells.
  • 0.3 ⁇ g/mL anti-CD3 antibody OKT3 (Thermo, #16-0037-81) was coated on a 96-well plate (Corning, #3599) at 100 ⁇ L/well.
  • the density of human T cells isolated from human PBMC using a T cell sorting kit (Miltenyi, #130-096-535) was adjusted to 2 ⁇ 10 6 cells/mL, and the density of target cells was adjusted to 3 ⁇ 10 5 cells/mL, followed by seeding 50 ⁇ L/well of each of the two cell suspensions in a 96-well plate.
  • the 96 well plate was placed 37 °C, 5% CO 2 incubator for 3 days.
  • the supernatants after 48 hours and 72 hours of culture were collected respectively, and the IL-2 concentration in the supernatants after 48 hours was detected by IL-2 ELISA kit (Thermo, #88-7025-88), and the IL-2 concentration in the supernatants after 48 hours was detected by IFN- ⁇ ELISA kit. (Thermo, #88-7316-77)
  • the IFN- ⁇ concentration in the supernatant after 72 hours was measured.
  • the ELISA detection method refers to the relevant kit operation instructions. Data processing and graph analysis were performed using the software GraphPad Prism 8.
  • control molecules were the corresponding parental mAbs PR002408 and PR002067.
  • neither the anti-OX40 HCAb mAb PR002067 nor the anti-B7H4 IgG mAb PR002408 could activate T cells in the presence of CHOK1/hB7H4 cells highly expressing B7H4; the B7H4 ⁇ OX40 bispecific binding protein (PR004277, PR004276) can activate T cells and promote the production of cytokine IL-2, which indicates that the activation of T cells by B7H4 ⁇ OX40 is dependent on target cells. Furthermore, the T-cell activating ability of the molecule with Fab-HCAb structure (PR004277) was slightly stronger than that of the molecule with IgG-VH structure (PR004276).
  • Example 7.1.1 Obtaining fully human IgG antibody against BCMA
  • the anti-BCMA recombinant fully human IgG antibody PR000892 (see Table 6 for the sequence) used in this example is derived from Harbour H2L2 mice, and its discovery process and sequence are disclosed in invention patent CN111234020B.
  • the Fab of the anti-BCMA IgG antibody PR000892 and the VH of the anti-BCMA HCAb antibody PR004433 were used to construct the anti-BCMA ⁇ BCMA bispecific binding protein PR005744 with the Fab-HCAb structure described in Example 1.1.1 .
  • the molecular design of PR005744 is shown in Table 4, and the corresponding sequence number is shown in Table 7; the molecule was prepared and analyzed according to the method described in Example 2, and is summarized in Table 9.
  • the antigen binding protein PR005744 was tested for its ability to bind BCMA and its ability to internalize on BCMA high expressing cells NCI-H929 (ATCC, CRL-9068).
  • biofilm interferometry (BLI) technique was used to analyze the binding kinetics of BCMA-binding protein and BCMA using Octet Molecular Interaction Analyzer (ForteBio, model Octet Red96e).
  • the recombinant human BCMA-ECD-Fc fusion protein (ACRO Biosystems, #BC7-H82F0) was first biotinylated using a biotinylation kit (EZ-Link Sulfo-NHS-LC-Biotin, ThermoFisher, A39257) according to the instructions.
  • the sensor used in this experiment was the SA biosensor (ForteBio, #18-5019); the working buffer was 1 ⁇ Kinetic Buffer (diluted from 10 ⁇ Kinetic Buffer (ForteBio, #18-1105)) for affinity testing and dilution of antigen and binding protein; equilibration buffer was 1 ⁇ PBS buffer (diluted from 10 ⁇ PBS buffer (BBI Life Sciences, #E607016-0500)).
  • Two columns of SA sensors (8 sensors per column; first column referred to as reference SA sensors and second column as test SA sensors) were first equilibrated in equilibration buffer for 10 minutes. Then, the test SA sensor captured biotinylated BCMA, setting the capture height to 0.2 nm, while the reference SA sensor was immersed in the buffer for 30 seconds. The two arrays of sensors were then combined with the BCMA-binding protein to be tested at a gradient dilution; the concentration of the BCMA-binding protein to be tested was 10-2.5 nM in a two-fold gradient dilution and 0 nM. The sensor binds to the protein to be tested for 180 seconds and then dissociates for 800 seconds.
  • the results are shown in Table 13 and Figure 13.
  • the tetravalent PR005744 has a higher binding affinity (KD value) to BCMA than the bivalent PR004433; and PR005744 has a higher maximum response signal (Response) than PR004433.
  • the tetravalent binding protein (PR005744) has a similar or even higher binding capacity for BCMA than the bivalent binding protein (PR004433).
  • This example further utilizes the FACS method to study the internalization of BCMA-targeting antigen binding proteins mediated killing of cells expressing human BCMA.
  • NCI-H929 (ATCC, CRL-9068) cells were seeded into a 96-well plate (Beyotime, #FT018) at 2 ⁇ 10 5 cells/well; then 200 nM of the antigen-binding protein to be tested diluted with FACS buffer was added Then, incubate at 4°C for 1 hour; then, take samples and incubate at 37°C for different times (eg, 30 minutes, 1 hour, 2 hours, and 4 hours); then, centrifuge and resuspend cells, add fluorescent secondary antibodies (Jackson ImmunoResearch , #109-545-098) and then incubated at 4°C for 30 minutes.
  • fluorescent secondary antibodies Jackson ImmunoResearch , #109-545-098
  • PR005744 was significantly more internalized than PR004433 in NCI-H929 cells; it could internalize more than 60% of BCMA within 30 minutes.
  • HER2 ⁇ CTLA4 can be enriched in tumor tissues with high expression of HER2, specifically release the inhibitory signal of CTLA4 in the tumor microenvironment to activate T cells, and reduce the toxic and side effects caused by non-specific activation of CTLA4 mAb in the peripheral system.
  • a number of molecules with Fab-HCAb structures containing different linking peptides were constructed to study the effect of linking peptides on the molecular structure of Fab-HCAb.
  • the anti-HER2 IgG antibody trastuzumab (protein number PR000210) was used, and its corresponding amino acid sequence was obtained from the IMGT database, and the sequence is shown in Table 6.
  • the Fab of the anti-HER2 IgG antibody PR000210 (trastuzumab analog), and the VH of the anti-CTLA4 HCAb antibody PR000184 were used to construct an anti-HER2 ⁇ CTLA4 bilayer having the Fab-HCAb structure described in Example 1.1.1 Specific binding proteins PR000305, PR000653, PR000654, PR000655 and PR000706. Its molecular design is shown in Table 4, and the corresponding sequence number is shown in Table 7; These bispecific binding protein molecules have a similar structure, the antigen binding domains Fab and VH are the same, the subtle difference lies in the different first linker peptide (between Fab and VH) and second linker peptide (VH and CH2 between) sequence.
  • This example utilizes these molecules to study the effect of different linker peptides on the molecular structure of Fab-HCAb.
  • flow cytometry FACS was used to test the binding ability of the binding protein to the tumor cell line SK-BR-3 (ATCC, HTB-30) highly expressing human HER2.
  • SK-BR-3 cells were digested and resuspended in complete medium, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL; then 100 ⁇ L cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), 4° C. Centrifuge for 5 minutes and discard the supernatant.
  • binding protein 100 ⁇ L/well of binding protein was added with a 5-fold concentration gradient dilution with a maximum final concentration of 100 nM, with a total of 8 concentrations, and mixed well; hIgG1 iso (CrownBio, #C0001) was used as an isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. After that, centrifuge at 4°C for 5 minutes, and discard the supernatant; then add pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) at 200 ⁇ L/well to rinse the cells twice, and then centrifuge at 500g for 5 minutes at 4°C , discard the supernatant.
  • FACS buffer PBS buffer containing 0.5% BSA
  • fluorescent secondary antibody Goat human IgG(H+L) Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution
  • Thermo Alexa Fluor 488 conjunction, Thermo, #A11013, 1:1000 dilution
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the antibody to the target cell and the EC50 value were obtained through four-parameter nonlinear fitting.
  • the positive control molecule is the anti-HER2 monoclonal antibody PR000210 (trastuzumab analog), which is also the parent monoclonal antibody at the HER2 end of HER2 ⁇ CTLA4.
  • the bispecific binding proteins of the Fab-HCAb structure (PR000305, PR000653, PR000654, PR000655 and PR000706) were comparable in their ability to bind to HER2 as the parental mAb PR000210, as reflected in nearly identical EC50 values and MFI maxima. This shows that the Fab end of the Fab-HCAb structure can well retain its corresponding target binding ability.
  • flow cytometry FACS was used to test the binding ability of the binding protein to cells such as CHO-K1 cell line CHO-K1/hCTLA4 (Ruizhi Chemical) that highly expresses human CTLA4.
  • CHO-K1/hCTLA4 cells were digested and resuspended with F12K medium; the cell density was adjusted to 2 ⁇ 10 6 cells/mL.
  • CHO-K1/hCTLA4 cells were seeded in a 96-well V-bottom plate (Corning, #3894) at 100 ⁇ L/well, centrifuged at 4° C. for 5 minutes, and the supernatant was discarded.
  • binding protein 100 ⁇ L/well of binding protein was added with a 5-fold concentration gradient dilution with a maximum final concentration of 300 nM, with a total of 8 concentrations, and mixed well; hIgG1 iso (CrownBio, #C0001) was used as an isotype control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour.
  • 100 ⁇ L/well of pre-cooled FACS buffer PBS buffer containing 0.5% BSA
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the binding curve of the antibody to the target cell and the EC50 value were obtained through four-parameter nonlinear fitting.
  • the positive control molecule is the anti-CTLA4 HCAb monoclonal antibody PR000184, which is also the parental monoclonal antibody at the CTLA4 end of HER2 ⁇ CTLA4.
  • the bispecific binding proteins of the Fab-HCAb structure can all bind CTLA4.
  • These molecules have similar structures and the same VH sequence at the CTLA4 end, with subtle differences in the different first linker peptides and the Fc-linked hinge region; thus these molecules have very similar abilities to bind CTLA4. This indicates that the length or sequence of different linker peptides has little effect on the VH of the binding domain in the Fab-HCAb structure.
  • these molecules bound CTLA4 with EC50 values similar to or only 1.5-3 times weaker than that of the parental mAb PR000184, but the maximal binding signal (MFI max) on FACS was lower than that of the parental mAb PR000184.
  • MFI max maximal binding signal
  • the Fab domain may have a "shielding" effect on the VH domain of HCAb, so that the Fab-HCAb molecule may preferentially bind to the target recognized by the Fab domain. , and only then will cause the binding of the VH domain.
  • This sequence of binding and the difference in binding force of different targets can be applied to the needs of some special application scenarios.
  • the recommended initial dose of anti-HER2 monoclonal antibody trastuzumab is 4 mg/kg in the treatment of breast cancer and 8 mg/kg in the treatment of gastric cancer; while the recommended initial dose of anti-CTLA4 monoclonal antibody ipilimumab in the treatment of melanoma is 3 mg/kg , lower doses in combination therapy.
  • the activity of the HER2 end of HER2 ⁇ CTLA4 of the Fab-HCAb structure is almost equivalent to that of its parent monoclonal antibody, but the activity of its CTLA4 end is relatively attenuated, so it can be used to achieve low and medium doses of CTLA4 inhibitors in clinical practice.
  • HER2 ⁇ CTLA4 can preferentially bind to HER2, enriching it in tumor tissues with high HER2 expression, thereby reducing the toxic and side effects caused by non-specific activation of T cells by CTLA4 antibodies in the peripheral system.
  • test antibody molecule 6 female BALB/c or C57BL/6 mice weighing 18-22 grams were selected, and the test antibody molecule was administered intravenously at a dose of 5 mg/kg.
  • One group of 3 animals collected whole blood before administration and 15 minutes, 24 hours (1 day), 4 days, and 10 days after administration, and the other group of 3 animals only before administration and 5 days after administration.
  • Whole blood was collected at hours, days 2, 7, and 14.
  • Whole blood was allowed to stand for 30 minutes to clot, then centrifuged and isolated serum samples were frozen at -80°C until analysis.
  • ELISA method 1 Two ELISA methods were used to quantify drug concentrations in mouse serum.
  • ELISA method 2 the Fc end detection method, captures the antibody containing human Fc in mouse serum by goat anti-human Fc polyclonal antibody coated on a 96-well plate, and then adds HRP-labeled goat anti-human Fc secondary antibody to detect.
  • ELISA method 2 the functional domain detection method, captures the antibody that specifically recognizes the antigen in mouse serum by coating the PD-L1 protein in a 96-well plate, and then adds HRP-labeled goat anti-human Fc secondary antibody to detection.
  • NCA non-compartmental model
  • the molecule PR004270 of the Fab-HCAb structure has a serum half-life t 1/2 value similar to that of conventional IgG antibodies, and the PD-L1 end detection method shows that its t 1/2 value exceeds 10 days.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供一种结合蛋白，其包括蛋白功能区A和蛋白功能区B，两者靶向不同的抗原或抗原的不同表位；所述蛋白功能区A为Fab；所述蛋白功能区B为VH，所述结合蛋白还包括Fc同源二聚体；所述蛋白功能区A和蛋白功能区B的数量均为二个；所述结合蛋白为左右对称的结构；所述结合蛋白从N末端至C末端依次为蛋白功能区A、蛋白功能区B和Fc，其中所述蛋白功能区A与所述蛋白功能区B通过L1连接，所述蛋白功能区B与所述Fc通过L2连接。所述结合蛋白具有较小的分子量、较少的多肽链、更简单的结构；其具有通用性的结构，可适用于多种不同的靶点组合；相较于其他结构的双特异性结合蛋白能够更强的激活效应细胞的能力。

Description

Fab-HCAb结构的结合蛋白
本申请要求申请日为2020/06/30的中国专利申请202010618158.0的优先权、申请日为2020/06/30的中国专利申请202010630471.6的优先权、以及申请日为2020/12/08的中国专利申请202011423832.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，尤其涉及Fab-HCAb结构的结合蛋白及其制备和应用。
背景技术
抗体是免疫系统在抗原刺激下，由B细胞产生的、可与相应抗原发生特异性结合的免疫球蛋白(Immunoglobulin，Ig)。大多数物种的抗体的基本结构为呈现“Y”型的四聚体形式，包含两个完全相同的重链(H链)和两个完全相同的轻链(L链)，也称为“H2L2”。重链包括靠近N端的重链可变区(VH)和靠近C端的重链恒定区(CH)；轻链包括靠近N端的轻链可变区(VL)和靠近C端的轻链恒定区(CL)。IgG抗体的重链恒定区有3个结构域，分别为CH1、CH2和CH3；CH1和CH2之间还有一段铰链区(hinge)。抗体的可变区是其识别和结合抗原的主要部位；抗体的可变区结构域VH和VL以及恒定区结构域CH1和CL共同组成了抗原结合片段(antigen-binding fragment，Fab)。CH2和CH3组成了可结晶片段(fragment crystallizable，Fc)，是发挥抗体的效应功能以及影响抗体的血清半衰期的主要部位。
在骆驼科及鲨鱼科动物血清中还天然存在一种缺失轻链的重链抗体(heavy-chain antibody，HCAb)。来源于骆驼科的重链抗体与常规抗体相比，除了缺少轻链外，其重链可变区与铰链区之间没有CH1区，只含有一个重链可变区(VHH)和两个重链恒定结构域(CH2和CH3)；其基本结构为重链二聚体。骆驼科动物的重链抗体的VHH片段与常规抗体的VH特征不同，其单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，分子量只有约13KDa，因此也被称作纳米抗体(Nanobody)或单域抗体(single-domain antibody)。重链抗体或者其衍生的纳米抗体在分子影像、诊断试剂等方面有独特优势，但是其非人源属性及其潜在的免疫原性风险限制了它的治疗性用途，需要经过进一步的抗体工程改造(例如抗体人源化)才能使其满足用于临床治疗的要求。
由于人的抗体天然结构为“H2L2”，VH和VL的缔合保证了抗体的稳定性和可溶性；如果没有VL的存在，VH上的原本由VL保护住的疏水性基团将暴露于水性溶剂，使得 VH易于发生聚集，进而导致抗体可溶性变差；因此无法从天然来源中获得有功能的人源的重链抗体。Frank Grosveld等人提出了一种利用转基因动物获得全人源重链抗体的方法(专利申请WO2007/096779)。Frank Grosveld等人构建了一种转基因小鼠，其小鼠内源的抗体重链基因座和轻链基因座都被敲除或者失活，使其无法产生小鼠的抗体；然后将人源的抗体重链基因片段(V、D、J片段)转入该小鼠，利用该小鼠自身的重排和突变机制产生具有人源抗体基因序列的抗体，而且由于没有轻链，产生的抗体就是人源的重链抗体。该转基因小鼠体内可以利用在VDJ重排后引入基因突变和自然选择的过程，选择有利于VH溶解性的VDJ组合和突变，有效地改善VH的溶解性，因此该转基因小鼠体内可以产生天然不存在的人源的重链二聚体结构。从该转基因小鼠获得的全人源重链抗体及其衍生的全人源单域抗体都有广阔的应用前景。
双特异性抗体(bispecific antibodies)和多特异性抗体(multispecific antibodies)是一类在天然的单克隆抗体基础上通过蛋白质工程技术制备出的具有两种或者多种不同特异性抗原结合位点的人工抗体。天然的单克隆抗体是单特异性的，即只能识别和结合一种抗原；双特异性抗体可以结合两种不同的抗原或者同一个抗原上的不同表位；而多特异性抗体则可能识别更多的抗原。这使得双特异性抗体可以实现一些单特异性抗体无法实现的作用机制和功能效果，这极大地扩展了双特异性抗体的治疗性应用场景。近年来随着肿瘤免疫的兴起，双特异性抗体吸引了越来越多的注意力、技术和资金支持，成为治疗性抗体市场中增长最快的一个领域。
双特异性抗体的结构设计是非常重要的。天然存在的二价IgG抗体由两个相同的重链和两个相同的轻链组成，含有两个相同的抗原结合位点。双特异性抗体需要利用蛋白质工程技术等手段通过结构设计引入两个不同的抗原结合位点，由此产生的分子的多肽链来源于两个不同的重链和两个不同的轻链。因此，双特异性抗体开发的一个最主要的挑战就是链的错配问题，即怎样从超过10种的不同的重链和轻链的组合中获得具有正确的链组合的功能性的双特异性抗体。为了解决这一问题，科学家已经研发出多种开发策略和技术平台，通过引入不同设计特征或功能特性来提高所需目标产物的均质性和产量。采用对称结构是一种解决链的错配问题的策略。大多数对称结构采用“2+2”结构设计，也称“四价双特异性”对称结构(tetravalent bispecific symmetric structures)。由于其抗原结合域可能有不同的结构、朝向和位置，这些对称结构的分子在分子大小和药学性能上有较大差异。对称结构仍然有轻链错配的问题；AbbVie的DVD-Ig技术平台，EpimAb的FIT-Ig技术平台，WuXi Biologics的WuXiBody技术平台等都利用不同的策略来解决轻链错配的问题；Aptevo和MedImmune等公司则通过引入scFv结构来解决轻链错配的问题。但是，各种技术手段都有其局限性，例如，FIT-Ig等技术产生的双抗分子的分子量在 250KDa左右，较大的分子尺寸可能会影响其细胞内吞和组织穿透等能力；而scFv结构的引入可能会带来稳定性和溶解性的影响；而且许多技术平台产生的双抗分子有至少三条不同的多肽链，增加了分子的复杂度。
因此，仍然亟需开发新型的双特异性抗体分子结构，使其具有较简单和稳定的分子结构和优秀的药学性能，以满足快速开发和降低生产成本的需求。
重链抗体及其衍生的单域抗体在构建双特异甚至多特异抗体方面有其独特的优势。重链抗体的抗原结合结构域只有常规抗体的Fab的四分之一大小；而且没有轻链，避免了轻链错配的问题。所以，利用重链抗体及其衍生的单域抗体，可以构建分子量较小的、多肽链较少的、结构更简单的双特异甚至多特异抗体。而且，全人源重链抗体相较于骆驼科动物的重链抗体，在免疫原性和成药性方面更有优势。
发明内容
为克服现有技术中缺乏结构简单和稳定、且具备优异的药学性能的双特异性结合蛋白的缺陷，本发明提供了一种具有“Fab-HCAb结构”的双特异性结合蛋白及其制备方法和应用。所述“Fab-HCAb结构”具有较小的分子量、较少的多肽链、结构简单等特点，还具有与IgG抗体相似的Fc效应子功能、优秀的分子稳定性和药学性能等。
为解决上述技术问题，本发明的技术方案之一为：提供一种含有至少两个蛋白功能区的结合蛋白，其中，所述结合蛋白包括蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原或相同抗原的不同表位，其中所述蛋白功能区A为Fab结构，所述蛋白功能区B为VH结构；所述结合蛋白还包括Fc同源二聚体(含有至少一个Fc)；
其中所述蛋白功能区A的数量为二个，所述蛋白功能区B的数量为二个；所述结合蛋白为对称结构，所述对称结构为左右对称的结构；
所述结合蛋白从N末端至C末端依次为蛋白功能区A、蛋白功能区B和Fc，其中所述蛋白功能区A与所述蛋白功能区B通过第一连接肽(L1)连接，所述蛋白功能区B与所述Fc通过第二连接肽(L2)连接。
本发明所述的结合蛋白中，二个的所述蛋白功能区B与所述Fc形成对称的单链抗体的二聚体形式，并在所述单链抗体的二聚体的N末端上连接了所述蛋白功能区A，此时所述蛋白功能区A可以以其CH1(例如参见图1，结构(2))或CL(例如参见图1，结构(1))与所述蛋白功能区B的N末端连接。
本发明中，所述的结合蛋白可为四价结合蛋白，所述结合蛋白例如具有如图1中结构(1)或(2)所示的结构；所述结合蛋白具有两条不同的多肽链。
较佳地，所述结合蛋白具有四条多肽链，分别为两条相同的短链(或称“多肽链1”)和两条相同的长链(或称“多肽链2”)，其中，(1)所述短链(或称“多肽链1”)从N末端至C末端依次包括VH_A-CH1，所述长链(或称“多肽链2”)从N末端至C末端依次包括VL_A-CL-L1-VH_B-L2-CH2-CH3；或(2)所述短链(或称“多肽链1”)从N末端至C末端依次包括VL_A-CL，所述长链(或称“多肽链2”)从N末端至C末端依次包括VH_A-CH1-L1-VH_B-L2-CH2-CH3。在结构(1)中所述蛋白功能区A以其CL的C末端与所述蛋白功能区B的N末端连接，蛋白功能区A的VL_A和蛋白功能区B的VH_B融合在同一条多肽链上，相对于结构(2)来说更加能够避免VL_A和VH_B的缔合产生的错配副产物。
本文中VL、VH、CL、CH的含义均为本领域常规，分别代表轻链可变区、重链可变区、轻链恒定区和重链恒定区，其中CH包括CH1、CH2和CH3，分别是重链恒定区的第一、第二和第三结构域；所述的CL是轻链恒定区结构域；_A与_B分别代表该功能区为蛋白功能区A或蛋白功能区B或其组成(即，VH_A代表蛋白功能区A的重链可变区，VH_B代表蛋白功能区B的重链可变区，VL_A代表蛋白功能区A的轻链可变区)；“-”代表联结不同结构区的多肽键或用来分隔不同结构区；C末端即肽链的羧基末端(也可写成“C’”)，N末端即肽链的氨基末端(也可写成“N’”)。上述不同蛋白功能区融合在同一条多肽链上，可以避免错配副产物。在一些实施方案中，L1和L2可以是相同的序列。在另一些实施方案中，L1和L2可以是不同的序列。当所述L1和/或L2为“-”时，连接肽的长度为0。较佳地，所述L1(第一连接肽)和L2(第二连接肽)独立地可为例如“-”、GS或如SEQ ID NOs：161-182的氨基酸序列所示。在一些实施方案中，所述L1的长度可优选为0、或如SEQ ID NOs：163、164或167的氨基酸序列所示。在一些实施方案中，所述L2可优选如SEQ ID NOs：169、178或179的氨基酸序列所示。在一些实施方案中，所述L1和L2分别如SEQ ID NO：167和SEQ ID NO：179的氨基酸序列所示。在一些实施方案中，所述L1的长度为0，所述L2如SEQ ID NO：178的氨基酸序列所示。在一些实施方案中，所述L1的长度为0，所述L2如SEQ ID NO：179的氨基酸序列所示。在一些实施方案中，所述L1和L2分别如SEQ ID NO：163和SEQ ID NO：178的氨基酸序列所示。在一些实施方案中，所述L1和L2分别如SEQ ID NO：164和SEQ ID NO：178的氨基酸序列所示。在一些实施方案中，所述L1和L2分别如SEQ ID NO：167和SEQ ID NO：178的氨基酸序列所示。在一些实施方案中，所述L1和L2分别如SEQ ID NO：163和SEQ ID NO：169的氨基酸序列所示。
在一些具体的实施例中，所述蛋白功能区A又称作针对第一抗原的抗体A或第一抗原结合结构域；所述蛋白功能区B又称作针对第二抗原的抗体B或第二抗原结合结构域。
在一些具体的实施方式中，所述的“Fab-HCAb结构”的双特异性结合蛋白含有至少一个来源于人源重链抗体的重链可变区结构域VH，并且能够结合两个或更多个抗原，或同一抗原的两个或更多个表位，或同一表位的两个或更多个拷贝。
在一些具体的实施方式中，所述的“Fab-HCAb结构”的双特异性结合蛋白含有的重链恒定区可以是优选为人IgG1、人IgG2、人IgG3或人IgG4的重链恒定区或其突变；所述突变优选自C220S、N297A、L234A、L235A、G237A和P329G中的一种或多种突变，所述突变位点使用EU编号规则。例如所述重链恒定区可以包括L234A、L235A、G237A、N297A或P329G中的一个、两个或三个突变，例如包含L234A和L235A的突变组合(LALA)或包含L234A、L235A和P329G的突变组合(AAG)或L234A、L235A和G237A的突变组合(AAA)等等。
在一些具体的实施例中，所述抗原选自PD-L1、HER2、B7H4、CTLA4、OX40、4-1BB和BCMA中的一种或多种。所述结合蛋白含有至少两个蛋白功能区即蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B是独立地来源于PD-L1抗体或其抗原结合片段、HER2抗体或其抗原结合片段、B7H4抗体或其抗原结合片段、CTLA4抗体或其抗原结合片段、OX40抗体或其抗原结合片段、4-1BB抗体或其抗原结合片段、和BCMA抗体或其抗原结合片段中的一个或多个。较佳地，所述蛋白功能区A为来源于PD-L1抗体或其抗原结合片段、HER2抗体或其抗原结合片段、B7H4抗体或其抗原结合片段或BCMA抗体或其抗原结合片段的Fab，和/或，所述蛋白功能区B为来源于CTLA4抗体或其抗原结合片段、4-1BB抗体或其抗原结合片段、OX40抗体或其抗原结合片段或BCMA抗体或其抗原结合片段的VH。更佳地，所述结合蛋白中：所述蛋白功能区A为来源于HER2抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于CTLA4抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于PD-L1抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于4-1BB抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于B7H4抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于4-1BB抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于B7H4抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于OX40抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于BCMA抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于BCMA抗体或其抗原结合片段的VH。
在一些具体的实施例中，所述的PD-L1抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：75、85和97所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、32和54所示。所列CDR的氨基酸序列按照Chothia定义规则 所示。
在一些具体的实施例中，所述的B7H4抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：78、83和100所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：15、37和59所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的4-1BB抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：73、83和95所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：11、30和52所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的4-1BB抗体或其抗原结合片段包含重链可变区(VH)；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：14、35和57所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的OX40抗体或其抗原结合片段包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、36和58所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的BCMA抗体或其抗原结合片段包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：17、39和61所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的BCMA抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：77、87和99所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、34和56所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的CTLA4抗体或其抗原结合片段包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：10、29和51所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述的HER2抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：74、84和96所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：12、31和53所示。所列CDR的氨基酸序列按照Chothia定义规则 所示。
在一些具体的实施例中，所述的PD-L1抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：118所示的氨基酸序列，所述VH包括如SEQ ID NO：108所示的氨基酸序列。
在一些具体的实施例中，所述的B7H4抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：121所示的氨基酸序列，所述VH包括如SEQ ID NO：113所示的氨基酸序列。
在一些具体的实施例中，所述的4-1BB抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：116所示的氨基酸序列，所述VH包括如SEQ ID NO：106所示的氨基酸序列。
在一些具体的实施例中，所述的4-1BB抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：111所示的氨基酸序列。
在一些具体的实施例中，所述的OX40抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：112所示的氨基酸序列。
在一些具体的实施例中，所述的BCMA抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：115所示的氨基酸序列。
在一些具体的实施例中，所述的BCMA抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：120所示的氨基酸序列，所述VH包括如SEQ ID NO：110所示的氨基酸序列。
在一些具体的实施例中，所述的CTLA4抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：105所示的氨基酸序列。
在一些具体的实施例中，所述的HER2抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含如SEQ ID NO：117所示的氨基酸序列，所述VH包含如SEQ ID NO：107所示的氨基酸序列。
在一些具体的实施例中，所述的PD-L1抗体或其抗原结合片段包含序列如SEQ ID NO：136所示的轻链和序列如SEQ ID NO：126所示的重链。
在一些具体的实施例中，所述的B7H4抗体或其抗原结合片段包含序列如SEQ ID NO：139所示的轻链和序列如SEQ ID NO：131所示的重链。
在一些具体的实施例中，所述的4-1BB抗体或其抗原结合片段包含序列如SEQ ID NO：134所示的轻链和序列如SEQ ID NO：124所示的重链。
在一些具体的实施例中，所述的4-1BB抗体或其抗原结合片段包含序列如SEQ ID  NO：129所示的重链。
在一些具体的实施例中，所述的OX40抗体或其抗原结合片段包含序列如SEQ ID NO：130所示的重链。
在一些具体的实施例中，所述的BCMA抗体或其抗原结合片段包含序列如SEQ ID NO：133所示的重链。
在一些具体的实施例中，所述的BCMA抗体或其抗原结合片段包含序列如SEQ ID NO：138所示的轻链和序列如SEQ ID NO：128所示的重链。
在一些具体的实施例中，所述的CTLA4抗体或其抗原结合片段包含序列如SEQ ID NO：123所示的重链。
在一些具体的实施例中，所述的HER2抗体或其抗原结合片段包含序列如SEQ ID NO：135所示的轻链和序列如SEQ ID NO：125所示的重链。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：75、85和97所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、32和54所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：14、35和57所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：78、83和100所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：15、37和59所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：14、35和57所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：78、83和100所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：15、37和59所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、36和58所示。所列CDR的 氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：77、87和99所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、34和56所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，所示氨基酸序列分别如SEQ ID NOs：17、39和61。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：74、84和96所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：12、31和53所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：10、29和51所示。所列CDR的氨基酸序列按照Chothia定义规则所示。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含氨基酸序列如SEQ ID NO：118所示的轻链可变区和氨基酸序列如SEQ ID NO：108所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：111所示的重链可变区。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含氨基酸序列如SEQ ID NO：121所示的轻链可变区和氨基酸序列如SEQ ID NO：113所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：111所示的重链可变区。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含氨基酸序列如SEQ ID NO：121所示的轻链可变区和氨基酸序列如SEQ ID NO：113所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：112所示的重链可变区。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含氨基酸序列如SEQ ID NO：120所示的轻链可变区和氨基酸序列如SEQ ID NO：110所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：115所示的重链可变区。
在一些具体的实施例中，所述结合蛋白包含两种蛋白功能区：蛋白功能区A和蛋白功能区B。其中，所述蛋白功能区A包含氨基酸序列如SEQ ID NO：117所示的轻链可变区和氨基酸序列如SEQ ID NO：107所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：105所示的重链可变区。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：147所示的氨基酸序列；第二多肽链包括如SEQ ID NO：153所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：136所示的氨基酸序列；第二多肽链包括如SEQ ID NO：183所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：147所示的氨基酸序列；第二多肽链包括如SEQ ID NO：184所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：155所示的氨基酸序列；第二多肽链包括如SEQ ID NO：158所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：155所示的氨基酸序列；第二多肽链包括如SEQ ID NO：156所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：159所示的氨基酸序列；第二多肽链包括如SEQ ID NO：160所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：141所示的氨基酸序列；第二多肽链包括如SEQ ID NO：142所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：141所示的氨基酸序列；第二多肽链包括如SEQ ID NO：143所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：141所示的氨基酸序列；第二多肽链包括如SEQ ID NO：144所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：141所示的氨基酸序列；第二多肽链包括如SEQ ID NO：145所示的氨基酸序列。
在一些具体的实施例中，所述结合蛋白包含两个多肽链：第一多肽链和第二多肽链。其中，第一多肽链包括如SEQ ID NO：141所示的氨基酸序列；第二多肽链包括如SEQ ID NO：149所示的氨基酸序列。
本申请中，所述的CDR均可包含在所限序列的基础上进行突变的情形。所述突变为在所述的VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、VL CDR3的氨基酸序列的基础上分别具有3、2或1个氨基酸的插入、缺失或替换。本申请中，在类似“具有3、2或1个氨基酸的插入、缺失或替换”中“氨基酸突变”是指相较于原氨基酸序列而言，变体的序列存在氨基酸的突变，包括在原氨基酸序列的基础上发生氨基酸的插入、缺失或替换。示例性的解释是对CDR的突变可以包含3个、2个或1个氨基酸的突变，这些CDR之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如可以是对CDR1进行1个氨基酸的突变，对CDR2和CDR3不进行氨基酸突变。
本申请中，所述的VH、VL或所述的多肽链均可包含在所限定的序列的基础上进行突变的情形。所述突变为所限定的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所限定的氨基酸序列具有至少85％序列同一性，并保持或改善了所述抗体或其抗原结合片段、结合蛋白的结合活性；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
为了解决上述技术问题，本发明第二方面提供了一种分离的核酸，其编码如本发明第一方面所述的结合蛋白。
为了解决上述技术问题，本发明第三方面提供了一种重组表达载体，其包含如本发明第二方面所述的分离的核酸。较佳地，所述表达载体包含真核细胞表达载体和/或原核细胞表达载体。
为了解决上述技术问题，本发明第四方面提供了一种转化体，其包含如本发明第二方面所述的分离的核酸或如本发明第三方面所述的重组表达载体。较佳地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
为了解决上述技术问题，本发明第五方面提供了一种结合蛋白的制备方法，其包含 培养如本发明第四方面所述的转化体，从培养物中获得结合蛋白。
为了解决上述技术问题，本发明第六方面提供了一种药物组合物，所述药物组合物包含如本发明第一方面所述的结合蛋白，以及药学上可接受的载体。较佳地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分。
为了解决上述技术问题，本发明第七方面提供了一种试剂盒，其包括如本发明第一方面所述的结合蛋白和/或如本发明第六方面所述的药物组合物。
较佳地，所述试剂盒还包括(i)施用结合蛋白或药物组合物的装置；和/或(ii)使用说明。
为了解决上述技术问题，本发明第八方面提供了一种套装药盒，所述套装药盒包括药盒一和药盒二，所述药盒一包括如本发明第一方面所述的结合蛋白和/或如本发明第六方面所述的药物组合物，所述药盒二包括其它抗体或药物组合物。
为了解决上述技术问题，本发明第九方面提供了一种给药装置，所述给药装置包括如本发明第一方面所述的结合蛋白和/或如本发明第六方面所述的药物组合物。
较佳地，所述给药装置还包括容纳或将所述合蛋白和/或所述药物组合物施用于受试者的部件，例如注射器、输液装置或植入式给药装置。
为了解决上述技术问题，本发明第十方面提供了一种如本发明第一方面所述的结合蛋白、如本发明第六方面所述的药物组合物、如本发明第七方面所述的试剂盒、如本发明第八方面所述的套装药盒、和/或如本发明第九方面所述的给药装置在制备诊断、预防和/或治疗癌症或其他疾病的药物中的应用。
较佳地，所述的癌症选自乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤和多发性骨髓瘤中的一种或多种。
为了解决上述技术问题，本发明第十一方面提供了一种体外或体内检测特异性抗原的方法，其包括使用如本发明第一方面所述的结合蛋白和/或如本发明第六方面所述的药物组合物进行检测。
为了解决上述技术问题，本发明第十二方面提供了如本发明第一方面所述的结合蛋白、如本发明第六方面所述的药物组合物、如本发明第七方面所述的试剂盒、如本发明第八方面所述的套装药盒、和/或如本发明第九方面所述的给药装置在诊断、预防和/或治疗癌症或其他疾病中的应用。
较佳地，所述的癌症选自乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤和多发性骨髓瘤中的一种或多种。
为了解决上述技术问题，本发明第十三方面提供了一种诊断、预防和/或治疗癌症或 其他疾病的方法，所述方法包括向有需要的患者施用如本发明第一方面所述的结合蛋白、如本发明第六方面所述的药物组合物、如本发明第七方面所述的试剂盒、如本发明第八方面所述的套装药盒、和/或如本发明第九方面所述的给药装置的步骤；
较佳地，所述的癌症选自乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤和多发性骨髓瘤中的一种或多种。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明提供了利用重链抗体(HCAb)和常规抗体的抗原结合区Fab所构建的具有Fab-HCAb结构的双特异性结合蛋白。本发明中Fab-HCAb结构的双特异性结合蛋白分子具有简单的和通用性的结构，可适用于多种不同的靶点组合；其具有较小的分子量、较少的多肽链、结构简单等特点，还具有与IgG抗体相似的Fc效应子功能、优秀的分子稳定性和药学性能等。而且，相较于现有的具有其他结构的双特异性结合蛋白更有优势。
在某一较佳地实施例中，Fab-HCAb结构的分子相较于FIT-Ig结构、VH-IgG结构或IgG-VH结构的分子有如下一个或多个优势：
(1)Fab-HCAb结构的分子量相对较小，只有两种不同的多肽链，结构更简单，几乎没有多肽链的错配；
(2)Fab-HCAb结构有更优的靶点结合能力；
(3)Fab-HCAb结构的第一结合结构域(Fab)和第二结合结构域(VH)之间的距离更有利于靶细胞(例如，肿瘤细胞)和效应细胞(例如，T细胞)之间相互作用形成免疫突触以进一步促进效应细胞的激活；
(4)Fab-HCAb结构有更强的效应细胞激活能力；
(5)Fab-HCAb结构的分子中可以无需额外的连接肽，减少因连接肽被剪切的风险。
(6)Fab-HCAb结构更加紧凑，其两个第二结合结构域(VH)之间的距离较近，在某些情形下更利于靶点的成簇和多聚化；
(7)Fab-HCAb结构可能会优先结合Fab结构域识别的靶点，之后才会引起VH结构域的结合，不同靶点结合的顺序以及结合力的差异可以适用于一些特殊的应用场景的需求，例如TAA×4-1BB的Fab-HCAb可以优先结合肿瘤靶点。
附图说明
图1为分子结构示意图。
图2显示了PD-L1×4-1BB分子结合人4-1BB细胞CHO-K1/hu 4-1BB的活性。
图3显示了PD-L1×4-1BB分子结合人PD-L1细胞CHO-K1/hPD-L1的活性。
图4显示了PD-L1×4-1BB分子在混合淋巴细胞反应(MLR)实验中激活T细胞：(A)IL-2释放水平；(B)IFN-γ释放水平。
图5显示了B7H4×4-1BB分子结合人4-1BB细胞CHO-K1/hu 4-1BB的活性。
图6显示了B7H4×4-1BB分子结合肿瘤细胞SK-BR-3的活性。
图7显示了B7H4×4-1BB分子通过SK-BR-3细胞介导T细胞特异性激活。
图8显示了PD-L1×4-1BB分子通过CHO-K1/hPD-L1细胞介导T细胞特异性激活。
图9显示了B7H4×OX40分子结合人OX40细胞CHO-K1/hu OX40的活性。
图10显示了B7H4×OX40分子结合肿瘤细胞SK-BR-3的活性。
图11显示了B7H4×OX40分子通过人B7H4细胞CHO-K1/hB7H4介导T细胞特异性激活。
图12显示了BCMA结合蛋白在NCI-H929细胞上的内化。
图13显示了用BLI方法测定BCMA结合蛋白与BCMA的亲和力：(A)重链抗体PR004433；(B)Fab-HCAb结构的双特异性结合蛋白PR005744。
图14显示了HER2×CTLA4分子结合肿瘤细胞SK-BR-3的活性。
图15显示了HER2×CTLA4分子结合人CTLA4细胞CHO-K1/hCTLA4的活性。
图16显示了Fab-HCAb结构的分子PR004270在小鼠体内的药代动力学。
图17显示了预测出的Fab-HCAb结构：(A)Fab-HCAb的三维结构模型，A1和A2是Fab端的抗原结合位点，B1和B2是VH端的抗原结合位点；(B)Fab-HCAb结构处于最为伸展状态时，不同抗原结合位点之间的相对距离；(C)FIT-Ig结构处于最为伸展状态时，不同抗原结合位点之间的相对距离。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“结合蛋白”或者“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在 称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR-1、CDR1、FR-2、CDR2、FR-3、CDR3和FR-4。重链和轻链的可变区含有与抗原相互作用的结合结构域。VH的三个CDR分别表示为HCDR1、HCDR2和HCDR3，也可表示为VH CDR1、VH CDR2和VH CDR3；VL的三个CDR分别表示为LCDR1、LCDR2和LCDR3，也可表示为VL CDR1、VL CDR2和VL CDR3。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，所述CDR的氨基酸序列均是按照Chothia定义规则所示出的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273：927-48，1997)。在本发明的技术方案中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则来确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见下表。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表-I本申请抗体CDR定义方法
  Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列； H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。本领域技术人员应当知晓的是，在用Chothia编码CDR时，有些位置会有插入位点的情况(可参见http：//bioinf.org.uk/abs/)。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本发明使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因全部或部分转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术等。
在本申请中，术语“特异性结合”通常是指抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“Fab”通常指常规抗体(例如IgG)中与抗原结合的部分，包括抗体的重链可变区VH、轻链可变区VL和重链恒定区结构域CH1以及轻链恒定区CL。在常规抗体中，VH的C端与CH1的N端联结形成重链Fd片段，VL的C端与CL的N端联结形成轻链，CH1的C端又进一步与重链的铰链区和其他恒定区结构域联结形成重链。在一些实施例中，“Fab”也指Fab的变体结构。例如，在某些实施例中，VH的C端与CL的N端联结形成一个多肽链，VL的C端与CH1的N端联结形成另一个多肽链，形成Fab(cross VH/VL)的结构；在某些实施例中，Fab的CH1不与铰链区联结，而是CL的C端与重链的铰链区联结，形成Fab(cross Fd/LC)的结构。
在本申请中，术语“VH”通常指抗体的重链可变区VH结构域，即可以是人或者其他动物的常规抗体(H2L2结构)的重链可变区VH，也可以是骆驼科等动物的重链抗体(HCAb结构)的重链可变区VHH，还可以是利用Harbour HCAb转基因小鼠产生的全 人源重链抗体(HCAb结构)的重链可变区VH。
在本申请中，术语“抗原结合片段”通常指任何可以与抗原特异结合的蛋白功能区域，既可以是“Fab”，也可以是“VH”，还可以是其他抗原结合形式(例如脂质运载蛋白(1ipocalins)、神经细胞粘附分子(NCAM)、纤维结合蛋白(fibronectin)、锚蛋白重复片段蛋白(DARPins)等衍生蛋白结构)。
在本申请中，术语“Fab-HCAb结构”即为表1和图1中的如结构(1)和结构(2)所示的结构。该结构包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2，也称长链，从氨基末端到羧基末端，其包含VL_A-CL-L1-VH_B-L2-CH2-CH3。或者，该结构还可以包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-L1-VH_B-L2-CH2-CH3。其中，VH_A和VL_A分别为常规抗体A的重链可变区和轻链可变区，VH_B为重链抗体B的重链可变区，CL是轻链恒定区结构域，CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域，L1和L2是连接肽。在某些实施例中，L1的长度可以为0。在某些实施例中，L2可以是IgG的铰链区或者铰链区衍生的连接肽序列，或是表2中所列序列。在某些实施例中，“Fab-HCAb结构”特指结构(1)的形式。
在本申请中，术语“肿瘤抗原”(tumor antigen)即可以是肿瘤特异性抗原(tumor specific antigen，TSA)还可以是肿瘤相关抗原(tumor associated antigen，TAA)。肿瘤特异性抗原是指肿瘤细胞所特有的、不存在于正常细胞或组织上的抗原。肿瘤相关抗原并非肿瘤细胞所特有，也存在于正常细胞或组织，但是肿瘤细胞增殖时高度表达。
在本申请中，术语“靶细胞”是指需要被清除掉的细胞，主要是肿瘤细胞，也可以是免疫抑制性细胞等。
在本申请中，术语“效应细胞”一般指在免疫应答中参与清除异物抗原和行使效应功能的免疫细胞。如浆细胞、细胞毒性T细胞、NK细胞等等。
在本申请中，术语“PD-L1”通常是指程序性死亡配体1蛋白、其功能变体和/或其功能片段。PD-L1也称为分化簇274(CD274)或B7同源物1(B7-H1)，并且是由(人类中)CD274基因编码的蛋白。PD-L1序列是本领域已知的。例如，示例性的全长人PD-L1蛋白的氨基酸序列可在NCBI登录号NP_054862或UniProt登录号Q9NZQ7下找到；示例性的全长食蟹猴PD-L1蛋白序列可在NCBI登录号XP_005581836或Uniprot登录号G7PSE7下找到。PD-L1主要表达在抗原呈递细胞以及多种肿瘤细胞。PD-L1与PD-1相互作用会下调T细胞的活性，减弱细胞因子的分泌，起到免疫抑制作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD- L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡，并进一步保护肿瘤细胞逃避免疫攻击。
在本申请中，术语“HER2”通常是指受体酪氨酸激酶erbB-2(也称为ERBB2)、其功能变体和/或其功能片段。HER2序列是本领域已知的。例如，示例性的全长的人HER2序列可以在Uniprot登录号P04626中找到；示例性的全长的食蟹猴HER2序列可以在NCBI登录号XP_005584091中找到。
在本申请中，术语“B7H4”通常是指含V-Set域T细胞激活抑制因子1(也称为VTCN1，B7h.5，B7S1，B7x)、其功能变体和/或其功能片段。B7H4序列是本领域已知的。例如，示例性的全长的人B7H4序列可以在Uniprot登录号Q7Z7D3中找到；示例性的全长的食蟹猴B7H4序列可以在NCBI登录号XP_005542249中找到；示例性的全长的小鼠B7H4序列可以在Uniprot登录号Q7TSP5中找到。B7-H4是一种隶属于B7/CD28超家族的跨膜蛋白。B7-H4蛋白表达于一些免疫细胞如单核细胞和树突状细胞，有可能参与T细胞的负调控免疫应答。此外，B7H4还在乳腺癌、卵巢癌、子宫内膜癌、非小细胞肺癌、肾癌等的肿瘤细胞表面上高表达，而在大多数正常组织中没有表达或者表达极低。B7-H4作为这些肿瘤的一个新兴靶点，近年来受到关注。抗B7-H4的抗体可以通过多种机制作用于肿瘤细胞，但是其研发方向主要集中在单克隆抗体上，目前尚无双特异性抗体疗法。
在本申请中，术语“4-1BB”通常是指肿瘤坏死因子受体超家族成员9(也称为CD137，TNFRSF9，4-1BBL受体)、其功能变体和/或其功能片段。4-1BB序列是本领域已知的。例如，示例性的全长的人4-1BB序列可以在Uniprot登录号Q07011中找到；示例性的全长的食蟹猴4-1BB序列可以在NCBI登录号XP_005544945中找到。4-1BB是一种隶属于TNF受体超家族的跨膜蛋白。4-1BB是在多种免疫细胞上表达的共刺激分子，为免疫活性的多功能调节剂。其诱导表达于活化的T细胞、NK细胞等免疫细胞。4-1BB通过其配体4-1BBL介导的三聚化来激活T细胞，促进细胞增殖和细胞因子释放。抗4-1BB的激动型抗体具有抑制肿瘤的功能，最早进入临床试验的4-1BB抗体是辉瑞的Utomilumab和百时美施贵宝(BMS)公司的Urelumab(BMS-663513)。Urelumab最初的临床结果发表于2008年，尽管在部分患者上观察到令人鼓舞的疗效，但数据显示Urelumab导致肝脏毒性，且与靶标和剂量有关。Utomilumab安全性更好，剂量可提高至10mg/kg，但治疗效果依然欠佳。4-1BB靶向性药物开发的核心问题是如何较为合适地通过4-1BB激活免疫细胞，在药效和安全性之间达到平衡。
在本申请中，术语“OX40”通常是指肿瘤坏死因子受体超家族成员4(也称为CD134，TNFRSF4，OX40L受体)、其功能变体和/或其功能片段。OX40序列是本领域已知的。例如，示例性的全长的人OX40序列可以在Uniprot登录号P43489中找到；示例性的全长 的食蟹猴OX40序列可以在NCBI登录号XP_005545179中找到。OX40是TNF受体超家族成员之一，参与增强T细胞受体触发的T细胞反应，是共刺激受体分子。它是一种50kD的跨膜蛋白。OX40瞬时表达在TCR刺激后的人CD4 +和CD8 +T细胞上。但在肿瘤部位，OX40在CD4 +T细胞上的表达比在CD8 +T细胞更高。因此，CD4 +和CD8 +T细胞是OX40定向免疫治疗癌症的药物的潜在靶标。OX40抗体一些临床前研究表明抗OX40的单抗通过促进MDSC的积累和生成Th2细胞因子而产生有害的免疫抑制副作用。
在本申请中，术语“BCMA”通常是指肿瘤坏死因子受体超家族成员17(也称为B-细胞成熟抗原，TNFRSF17，CD269)、其功能变体和/或其功能片段。BCMA序列是本领域已知的。例如，示例性的全长的人BCMA序列可以在Uniprot登录号Q02223中找到；示例性的全长的食蟹猴BCMA序列可以在NCBI登录号XP_005591343中找到。BCMA是一种属于TNF受体超家族的跨膜蛋白，其参与B细胞成熟、生长和存活。BCMA主要有两种配体：高亲和力配体APRIL以及低亲和力配体BAFF。BCMA在多发性骨髓瘤(MM)患者的恶性浆细胞中表达，支持多发性骨髓瘤细胞的生长和存活。多发性骨髓瘤是继非霍奇金淋巴瘤的血液系统第二大恶性肿瘤，约占血液系统恶性肿瘤的13％。作为多发性骨髓瘤的一个新兴靶点，BCMA抗体可以通过多种机制作用于MM细胞。
在本申请中，术语“CTLA4”通常是指细胞毒性T淋巴细胞相关蛋白-4(也称为CD152)、其功能变体和/或其功能片段。CTLA4序列是本领域已知的。例如，示例性的全长的人CTLA4序列可以在Uniprot登录号P16410中找到；示例性的全长的食蟹猴CTLA4序列可以在Uniprot登录号G7PL88中找到。CTLA4是T细胞上表达的负调控因子，它与抗原递呈细胞上的CD80或CD86结合后，在阻断CD28的共剌激信号同时，还会下调T细胞的活性，起到免疫抑制作用。通过阻断CTLA4与其配体的相互作用可以恢复T细胞的活性，增强抗肿瘤的能力。Ipilimumab单抗(商品名
Figure PCTCN2021102935-appb-000001
)是第一个获批上市的抗CTLA4单抗药物。Ipilimumab在晚期黑色素瘤的治疗上体现出较好的治疗效果，但是Ipilimumab也带来了较高的免疫相关副反应，这严重地影响了它的临床应用。Ipilimumab所表现出来的毒副作用大部分是CTLA4靶点相关的，在目前的PD-1/PD-L1抑制剂和CTLA4抑制剂的联合用药方案中，CTLA4抑制剂无论Ipilimumab或是Tremelimumab都通常选用较低剂量。为了降低CTLA4抑制剂的毒副作用，其中一种值得尝试的方法是将CTLA4抑制剂定向输送到肿瘤组织内部，使相关的T细胞介导的反应仅局限于肿瘤微环境内，而减少细胞因子释放综合征的风险。例如，利用识别肿瘤相关抗原(tumor-associated antigen)的抗体将CTLA4抑制剂重定向到特定的肿瘤微环境中，使其在肿瘤微环境中解除T细胞的免疫抑制信号，恢复T细胞的功能。
实施例
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1 基于重链抗体的双特异性结合蛋白
本实施例中的表1和图1列出了本发明申请所涉及的利用重链抗体(HCAb)及其衍生的单域抗体(sdAb)所构建的双特异性结合蛋白的结构。每一种结构会在下文进一步描述。在本发明申请中，所述Fab-HCAb结构即为表1和图1中的如结构(1)和结构(2)所示的结构，且优选结构为结构(1)。
在有些结构中，结构域和结构域之间用连接肽进行联结。在有些结构中，重链的Fc区域引入了氨基酸突变以改变其与Fc受体的结合进而改变相关的效应功能或者其他性能。表2列出了本申请的结构设计中可能用到的连接肽序列。
表1本申请所列举的多特异性结合蛋白分子结构
Figure PCTCN2021102935-appb-000002
表2连接肽序列
连接肽名字 长度 序列 SEQ ID NO
GS_2 2 GS  
GS_4 4 GSGS 161
GS_5 5 GGGGS 162
GS_7 7 GGGGSGS 163
GS_15 15 GGGGSGGGGSGGGGS 164
GS_20 20 GGGGSGGGGSGGGGSGGGGS 165
GS_25 25 GGGGSGGGGSGGGGSGGGGSGGGGS 166
H1_15 15 EPKSSDKTHTPPPPP 167
LH1 10 DKTHTCPPCP 168
G5-LH 15 GGGGGDKTHTCPPCP 169
H1_15-RT 17 EPKSSDKTHTPPPPPRT 170
L-GS_15-RT 18 LGGGGSGGGGSGGGGSRT 171
L-H1_15-RT 18 LEPKSSDKTHTPPPPPRT 172
KL-H1_15-RT 19 KLEPKSSDKTHTPPPPPRT 173
KL-H1_15-AS 19 KLEPKSSDKTHTPPPPPAS 174
RT-GS_5-KL 9 RTGGGGSKL 175
RT-GS_15-KL 19 RTGGGGSGGGGSGGGGSKL 176
RT-GS_25-KL 29 RTGGGGSGGGGSGGGGSGGGGSGGGGSKL 177
人IgG1铰链 15 EPKSCDKTHTCPPCP 178
人IgG1铰链(C220S) 15 EPKSSDKTHTCPPCP 179
人IgG2铰链 12 ERKCCVECPPCP 180
人IgG4铰链 12 ESKYGPPCPSCP 181
人IgG4铰链(S228P) 12 ESKYGPPCPPCP 182
实施例1.1 含有重链抗体VH结构域的双特异性结合蛋白结构
本发明提供了一种使用两种亲本单克隆抗体构建双特异性结合蛋白的方法：结合第一抗原的常规抗体A(例如，IgG抗体)和结合第二抗原的重链抗体B。
如图1中结构(1)-(4)所示，Fab端来源于常规抗体A，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区。VH端来源于重链抗体B，VH_B为重链抗体B的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。h是IgG抗体的铰链区或衍生序列，L或L1或L2是连接肽。
实施例1.1.1 结构(1)：Fab(CL)-VH-Fc
结构(1)的结合蛋白包含两条不同的多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2，也称长链，从氨基末端到羧基末端，其包含VL_A-CL-L1-VH_B-L2-CH2-CH3。在结构(1)中，抗体A的VL_A和重链抗体B的VH_B融合在同一条多肽链上，这样可以避免VL_A和VH_B的缔合产生的错配副产物。
多肽链2的VH_B经由连接肽L2联结到CH2；L2可以是IgG的铰链区或者铰链区衍生的连接肽序列，或是表2中所列序列，优选为人IgG1铰链或者人IgG1铰链(C220S) 或者G5-LH的序列。
在一个实施方案中，多肽链2的CL与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CL经由连接肽L1联结到VH_B；L1可以是表2中所列序列。
实施例1.1.2 结构(2)：Fab(CH1)-VH-Fc
结构(2)的结合蛋白包含两条不同的多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-L1-VH_B-L2-CH2-CH3。
多肽链2的VH_B经由连接肽L2联结到CH2；L2可以是IgG的铰链区或者铰链区衍生的连接肽序列，或是表2中所列序列，优选为人IgG1铰链或者人IgG1铰链(C220S)或者G5-LH的序列。
在一个实施方案中，多肽链2的CH1与VH_B直接融合联结，即L1的长度为0。在另一个实施方案中，多肽链2的CH1经由连接肽L1联结到VH_B；L1可以是表2中所列序列。
实施例1.1.3 结构(3)：IgG_HC-VH
结构(3)的结合蛋白包含两条不同的多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L-VH_B。
在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链2的CH3经由连接肽L联结到VH_B；L可以是表2中所列序列。
实施例1.1.4 结构(4)：VH-IgG_HC
结构(4)的结合蛋白包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_B-L-VH_A-CH1-h-CH2-CH3。
在一个实施方案中，多肽链2的VH_B与VH_A直接融合联结，即L的长度为0。在另一个实施方案中，多肽链2的VH_B经由连接肽L联结到VH_A；L可以是表2中所列序列。
实施例1.2 其他双特异性结合蛋白结构
实施例1.2.1 结构(5)：FIT-Ig
FIT-Ig结构的设计可以参考专利WO2015/103072A1，见图1的结构(5)所示。FIT-Ig结构的双抗分子可以构建自：结合第一抗原的常规抗体A和结合第二抗原的常规抗体B。
结构(5)的结合蛋白包含三条多肽链：多肽链1，从氨基末端到羧基末端，其包含VL_A-CL-L-VH_B-CH1-h-CH2-CH3；多肽链2，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链3，从氨基末端到羧基末端，其包含VL_B-CL。其中，VH_A和VL_A分别为抗体A的重链可变区和轻链可变区，VH_B和VL_B分别为抗体B的重链可变区和轻链可变区，CL是轻链恒定区结构域，CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域，h是IgG抗体的铰链区或衍生序列，L是连接肽。一般情况下，多肽链2和多肽链3的缔合会产生错配副产物VH_A-CH1/VL_B-CL。
在一个实施方案中，多肽链1的CL与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链1的CL经由连接肽L联结到VH_B；L可以是表2中所列序列。
实施例2 抗体的序列分析、表达纯化、和理化性质表征分析
实施例2.1 抗体的表达和纯化
本实施例介绍了利用哺乳动物宿主细胞(例如，人胚肾细胞HEK293或中国仓鼠卵巢细胞CHO及其衍生细胞)、瞬时转染表达和亲和捕获分离等技术来制备抗体的一般方法。本方法适用于含有Fc的目标抗体；目标抗体可以由一条或多条蛋白质多肽链组成；可以来源于一个或多个表达质粒。
将抗体多肽链的氨基酸序列通过密码子优化方法转换成核苷酸序列；合成编码的核苷酸序列并克隆到与宿主细胞兼容的表达载体上。将编码抗体多肽链的质粒按照特定比例同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到具有正确折叠和多肽链组装的重组抗体。具体地，将FreeStyle TM293-F细胞(Thermo，#R79007)在FreeStyle TMF17 Expression Medium培养基(Thermo，#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6-8x10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2x10 6细胞/ml。准备30ml培养的细胞。将编码抗体多肽链的质粒按照一定比例混合共计30μg质粒(质粒与细胞的比例为1μg∶1ml)溶解于1.5ml Opti-MEM减血清培养基(Thermo，#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences，#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中， 室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS pH7.4缓冲液平衡含有MabSelect TM(GE Healthcare，#71-5020-91)的重力柱(Bio-Rad，#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS缓冲液冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，随后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore，#UFC901024)浓缩换液至PBS缓冲液或者含有其他成分的缓冲液，得到纯化的重组抗体溶液。最后用NanoDrop(Thermo，NanoDrop TMOne)测定浓度，分装、存储备用。
实施例2.2 利用SEC-HPLC分析蛋白纯度和聚体
本实施例使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience，#08541，5μm，7.8mm×30em)连接到高压液相色谱仪HPLC(Agilent Technologies，Agilent 1260 Infinity II)，用PBS缓冲液室温下平衡至少1小时。适量蛋白样品(至少10μg)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用PBS缓冲液将样品以1.0ml/分钟的流速流过色谱柱，最长时间为25分钟。HPLC将生成分析报告，报告出样品内不同分子尺寸组份的滞留时间。
实施例3 构建具有Fab-HCAb结构和其他结构的双特异性结合蛋白
本实施例总结了本发明申请的各个实施例中所使用的IgG单抗和HCAb单抗以及衍生的双特异性结合蛋白。
IgG单抗和HCAb单抗的信息列于表3，其序列编号见表6，氨基酸序列见表11。
按照实施例1.1.1和图1(1)所述结构或实施例1.1.2和图1(2)所述结构设计具有Fab-HCAb结构的双特异性结合蛋白，其分子设计总结于表4，其序列编号见表7，氨基酸序列见表12；并按照实施例2所述方法制备蛋白样品并进行分析，总结于表9。
其他结构的双特异性结合蛋白的分子信息总结于表5，对应的结构编号即为实施例1和图1中的结构(3)、(4)或(5)；其序列编号见表7，氨基酸序列见表12；并按照实施例2所述方法制备蛋白样品并进行分析，总结于表10。
表8还列出了双特异性结合蛋白的蛋白功能区A(第一抗原结合结构域)和蛋白功能区B(第二抗原结合结构域)的对应的CDR序列的序列编号。
在有些结合蛋白的结构中，重链的Fc区域引入了氨基酸突变以改变其与Fc受体的结合进而改变相关的效应功能或者其他性能。例如，在表4和表5中，表中的突变位点代号是：AAG：(L234A，L235A，P329G)；LALA：(L234A，L235A)。
表3本申请所使用的对照分子和亲本单抗
蛋白编号 说明
PR000628 抗4-1BB单抗Urelumab类似物(hIgG4)
PR003475 抗OX40单抗Pogalizumab类似物(hIgG1)
PR000210 抗HER2单抗Trastuzumab类似物(hIgG1)
PR000265 抗PD-L1H2L2单抗91G3H5H3(D54E)，hIgG1(N297A)
PR002408 抗B7H4H2L2单抗80C8-2E9(H：G55A；L：N92Q)，hIgG1
PR000197 抗4-1BB H2L2单抗79B10G8D4，hIgG4
PR001760 抗4-1BB重链抗体1016P0011G10
PR002067 抗OX40重链抗体R1026P079E12
PR004433 抗BCMA重链抗体PR001046_R2_4G10
PR000892 抗BCMA H2L2单抗1005_21C11E1，hIgG1
PR000184 抗CTLA4重链抗体CL5v3
表4本申请中具有Fab-HCAb结构的双特异性结合蛋白
Figure PCTCN2021102935-appb-000003
表5本申请中其他结构的双特异性结合蛋白
Figure PCTCN2021102935-appb-000004
表6本申请中对照分子和亲本单抗的可变区和CDR的序列编号表
Figure PCTCN2021102935-appb-000005
表7本申请中双特异性结合蛋白的序列编号表
结构编号 蛋白编号 多肽链1 多肽链2 多肽链3
1 PR004270 147 153  
2 PR007163 136 183  
1 PR007164 147 184  
1 PR004279 155 158  
1 PR004277 155 156  
1 PR005744 159 160  
1 PR000305 141 142  
1 PR000653 141 143  
1 PR000654 141 144  
1 PR000655 141 145  
1 PR000706 141 149  
3 PR003335 139 150  
3 PR003550 136 151  
3 PR004276 139 154  
4 PR004268 136 152  
4 PR004278 139 157  
5 PR000701 148 147 146
表8本申请中双特异性结合蛋白的抗原结合结构域的CDR序列编号表
Figure PCTCN2021102935-appb-000006
Figure PCTCN2021102935-appb-000007
表9本申请中Fab-HCAb结构的双特异性结合蛋白的表达
Figure PCTCN2021102935-appb-000008
Figure PCTCN2021102935-appb-000009
表10本申请中其他结构的双特异性结合蛋白的表达
Figure PCTCN2021102935-appb-000010
表11本申请中对照分子和亲本单抗的氨基酸序列
Figure PCTCN2021102935-appb-000011
Figure PCTCN2021102935-appb-000012
Figure PCTCN2021102935-appb-000013
Figure PCTCN2021102935-appb-000014
Figure PCTCN2021102935-appb-000015
Figure PCTCN2021102935-appb-000016
表12本申请中双特异性结合蛋白的氨基酸序列
Figure PCTCN2021102935-appb-000017
Figure PCTCN2021102935-appb-000018
Figure PCTCN2021102935-appb-000019
Figure PCTCN2021102935-appb-000020
Figure PCTCN2021102935-appb-000021
Figure PCTCN2021102935-appb-000022
Figure PCTCN2021102935-appb-000023
Figure PCTCN2021102935-appb-000024
实施例4 PD-L1×4-1BB双特异性结合蛋白
在本实施例中，我们构建了靶向PD-L1和4-1BB的具有Fab-HCAb、IgG-VH、VH-IgG或FIT-Ig结构的双特异性结合蛋白PD-L1×4-1BB，通过一个或者多个作用机制来提高抗肿瘤效果和安全性。第一，PD-L1×4-1BB可以通过阻断PD-1/PD-L1信号通路来激活T细胞。第二，高表达于肿瘤细胞表面的PD-L1分子可以利用PD-L1×4-1BB促进T细胞表面的4-1BB分子的交联和三聚化并激活下游信号传导通路，进而促进T细胞的活化和增殖。第三，PD-L1×4-1BB介导的T细胞激活仅限于在肿瘤微环境内，这样可以避免类似Urelumab的单抗在正常组织中过度激活T细胞所带来的毒副作用。
实施例4.1 获得抗PD-L1的IgG抗体和抗4-1BB的IgG或HCAb抗体
实施例4.1.1 获得抗PD-L1的全人源IgG抗体
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人PD-L1蛋白(NovoProtein，#CM06)对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中PD-L1特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，鉴定出若干个特异识别PD-L1的单克隆抗体分子。对这些单克隆抗体进行进一步的鉴定，根据其对人PD-L1的结合能力、食蟹猴PD-L1的结合能力、抑制PD-L1与PD-1结合能力等参数，优选出数个候选抗体分子。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。
抗PD-L1的重组全人源IgG抗体PR000265的序列见表6。
实施例4.1.2 获得抗4-1BB的全人源IgG抗体
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基 因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人4-1BB-Fc融合蛋白(南京金斯瑞生物科技)对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中4-1BB特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，鉴定出若干个特异识别4-1BB的单克隆抗体分子。对这些单克隆抗体进行进一步的鉴定，根据其对人4-1BB的结合能力、食蟹猴4-1BB的结合能力、T细胞激活能力等参数，优选出数个候选抗体分子。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。
抗4-1BB的重组全人源IgG抗体PR000197的序列见表6。
实施例4.1.3 获得抗4-1BB的全人源HCAb抗体
Harbour HCAb小鼠(Harbour Antibodies BV，WO2010/109165A2)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生仅有重链的抗体，该抗体的分子量只有传统IgG抗体的一半。其产生的抗体仅具有人的抗体重链可变结构域和小鼠Fc恒定结构域。
用可溶的重组人4-1BB-Fc融合蛋白(睿智化学提供)或者过表达了人4-1BB的NIH-3T3细胞(睿智化学提供)对Harbour HCAb小鼠进行多轮免疫。当检测小鼠血清中4-1BB特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用小鼠浆细胞分选试剂盒(Miltenyi，#130-092-530)分选CD138阳性的浆细胞。用常规的分子生物学手段从浆细胞中扩增人VH基因，并将扩增的人VH基因片段构建到编码人IgG1抗体重链Fc序列的哺乳动物细胞表达质粒pCAG载体中。质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，得到全人源HCAb抗体上清。用FACS测试HCAb抗体上清与高表达人4-1BB的CHO-K1细胞CHO-K1/hu4-1BB的结合，鉴定出阳性HCAb抗体。对这些HCAb抗体进行进一步的鉴定，根据其对人4-1BB的结合能力、食蟹猴4-1BB的结合能力、T细胞激活能力等参数，优选出数个候选HCAb抗体分子。
抗4-1BB的重组全人源HCAb抗体PR001760的序列见表6。
实施例4.2 构建PD-L1×4-1BB双特异性结合蛋白
一方面，本实施例利用抗PD-L1的IgG抗体PR000265的Fab，和抗4-1BB的HCAb抗体PR001760的VH，来构建具有如实施例1.1.1所述Fab-HCAb结构(图1结构(1)：Fab(CL)-VH-Fc)的抗PD-L1×4-1BB的双特异性结合蛋白PR004270和PR007164；和构建具有如实施例1.1.2所述Fab-HCAb结构(图1结构(2)：Fab(CH1)-VH-Fc)的抗PD-L1×4-1BB的双特异性结合蛋白PR007163。PR004270、PR007163和PR007164的分 子设计见表4，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表9。如表9中所示，PR007164(结构(1))、PR004270的纯化后产量显著高于PR007163(结构(2))。
另一方面，本实施例还利用抗PD-L1的IgG抗体PR000265的Fab，和抗4-1BB的HCAb抗体PR001760的VH，来构建具有如实施例1.1.3所述IgG-VH结构的抗PD-L1×4-1BB的双特异性结合蛋白PR003550。PR003550的分子设计见表5，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表10。
另一方面，本实施例还利用抗PD-L1的IgG抗体PR000265的Fab，和抗4-1BB的HCAb抗体PR001760的VH，来构建具有如实施例1.1.4所述VH-IgG结构的抗PD-L1×4-1BB的双特异性结合蛋白PR004268。PR004268的分子设计见表5，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表10。
另一方面，本实施例还利用抗PD-L1的IgG抗体PR000265的Fab，和抗4-1BB的IgG抗体PR000197的Fab，来构建具有如实施例1.2.1所述FIT-Ig结构的抗PD-L1×4-1BB的双特异性结合蛋白PR000701。PR000701的分子设计见表5，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表10。
实施例4.3 结合4-1BB
本实施例利用流式细胞术FACS测试结合蛋白与高表达人4-1BB的CHO-K1细胞株CHO-K1/hu4-1BB(南京金斯瑞，M00538)细胞的结合能力。具体地，消化细胞并用完全培养基重悬；将细胞密度调整为2x10 6细胞/mL。接着将细胞以100μL/孔(2x10 5细胞/孔)接种于96孔V底板(Corning，#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的结合蛋白以100μL/孔加入96孔板并混合均匀，结合蛋白可以从最高终浓度为200nM按照3倍浓度梯度稀释的共12个浓度；hIgG1 iso(CrownBio，#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction，Thermo，#A11013，1∶1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合蛋白对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗4-1BB的单抗Urelumab(蛋白编号PR000628)或抗4-1BB的HCAb抗体PR001760。
图2中所示，PD-L1×4-1BB双特异性结合蛋白(PR004270，PR004268，PR003550)结合4-1BB的能力相当，且在MFI最大值上优于阳性对照Urelumab，并在EC50值上优于FIT-Ig结构的分子PR000701。PD-L1×4-1BB双特异性结合蛋白(PR007163，PR007164)结合4-1BB的能力与其亲本单抗PR001760的相当。
实施例4.4 结合PD-L1
本实施例利用流式细胞术FACS测试结合蛋白与高表达人PD-L1的CHO-K1细胞株CHO-K1/hPD-L1(南京金斯瑞，M00543)的结合能力。具体地，消化细胞并用完全培养基重悬；将细胞密度调整为1x10 6细胞/mL。接着将细胞以100μL/孔接种于96孔V底板(Corning，#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的结合蛋白以100μL/孔加入96孔板并混合均匀，结合蛋白可以从最高终浓度为200nM按照3倍浓度梯度稀释的共12个浓度；hIgG1 iso(CrownBio，#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction，Thermo，#A11013，1∶1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合蛋白对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗PD-L1的单抗PR000265，亦为PD-L1×4-1BB的PD-L1端的亲本单抗。
图3中所示，Fab-HCAb结构的分子(PR004270)和VH-IgG结构的分子(PR004268)结合PD-L1的能力与亲本单抗PR000265相似，其结合PD-L1的EC50值虽略弱于亲本单抗，但是结合的MFI最大值比亲本单抗更高。IgG-VH结构的分子(PR003550)结合PD-L1的能力与亲本单抗PR000265相似，且EC50值和MFI最大值略优于FIT-Ig结构的分子(PR000701)。Fab-HCAb结构的分子(PR007163，PR007164)结合PD-L1的能力与亲本单抗PR000265相当。
实施例4.5 混合淋巴细胞反应(MLR)
本实施例是利用混合淋巴细胞反应(MLR)来研究PD-L1×4-1BB双特异结合蛋白对T细胞的激活作用。
第一步，利用CD14磁珠(Meltenyi，#130-050-201)从第一供体PBMC细胞(妙通生物)中分离单核细胞(monocytes)；具体操作参照相关试剂盒说明书。然后加入50ng/mL重组人源IL-4(PeproTech，#200-02-A)和100ng/mL重组人源GM-CSF(PeproTech，#300-03-A)，于37℃诱导7天后，获得未成熟的树突状细胞(iDC细胞)。继续加入1μg/ml的脂多糖Lipopolysaccharide(LPS，Sigma，#L6529)，诱导24小时后，获得成熟的树突状细胞(mDC细胞)。第二步，利用T细胞分离试剂盒(Meltenyi，#130-096-535)从第二供体PBMC细胞(妙通生物)中分离得到T淋巴细胞。第三步，将获得的T细胞和mDC细胞按5∶1比例接种至96孔板(1×10 5/孔的T细胞和2×10 4/孔的mDC细胞)。随后以50μL/孔加入不同浓度的结合蛋白，其终浓度可以是(10nM，1nM)；3个复孔加样；hIgG1iso(CrownBio，#C0001)或者空白孔作为对照。于37℃，5％CO 2培养箱孵育5天。第四步，分别收集第4天和第5天的上清液，用IL-2ELISA试剂盒(Thermo，#88-7025-88)检测第4天的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo，#88-7316-77)检测第5天的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。应用软件GraphPad Prism 8进行数据处理和作图分析。
图4中所示，在MLR实验中，抗4-1BB单抗(PR001760)对T细胞的激活作用有限，产生细胞因子(IFN-γ，IL-2)的能力很弱；但是，抗PD-L1单抗(PR000265)有较明显的激活作用。另一方面，PD-L1×4-1BB双特异结合蛋白可以进一步提高T细胞的功能，优于抗PD-L1单抗。而且相较于FIT-Ig结构的分子(PR000701)，IgG-VH结构的分子(PR003550)和Fab-HCAb结构的分子(PR004270)能够刺激T细胞产生更多的细胞因子。
实施例4.6 靶细胞介导的T细胞的特异性激活
本实施例是为了研究PD-L1×4-1BB双特异结合蛋白在靶细胞的存在时通过结合4-1BB来激活T细胞的活性。靶细胞可以是高表达人PD-L1的细胞CHO-K1/hPD-L1(南京金斯瑞，M00543)；效应细胞可以是分离的人PBMC或者T细胞。
具体的，首先以100μL/孔将0.3μg/mL抗CD3抗体OKT3(Thermo，#16-0037-81)包板于96孔板(Corning，#3599)。接着，将人T细胞(从人PBMC中用T细胞分选试剂盒(Miltenyi，#130-096-535)分离得到)的密度调整为2x10 6细胞/mL，将靶细胞的密度调整为3x10 5细胞/mL，随后把两种细胞悬液各以50μL/孔接种于96孔板，最终效靶比 为20∶3。然后，以100μL/孔加入不同浓度的结合蛋白，其终浓度可以是(10nM，1nM)；2个复孔加样；hIgG1 iso(CrownBio，#C0001)和hIgG4 iso(CrownBio，#C0045)作为对照。将96孔板置于37℃，5％CO 2培养箱中孵育3天。分别收集培养48小时后和72小时后的上清液，用IL-2ELISA试剂盒(Thermo，#88-7025-88)检测48小时后的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo，#88-7316-77)检测72小时后的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。应用软件GraphPad Prism 8进行数据处理和作图分析。
图8中所示，在靶细胞CHO-K1/hPD-L1和T细胞混合的系统中，不依赖于交联的抗4-1BB单抗Urelumab可以激活T细胞释放IFN-γ；Fab-HCAb结构的分子(PR004270)具有最强的T细胞激活能力，其IFN-γ水平高于Urelumab和其他结构的双特异分子(如，PR003550，PR000701)。总体说来，T细胞激活能力排序：PR004270＞PR003550＞Urelumab＝PR000701＞PR004268。
实施例4.7 结构模拟
在实施例4.5和实施例4.6中，Fab-HCAb结构的分子(PR004270)相较于FIT-Ig结构的分子(PR000701)，显示出更强的T细胞激活能力。为了进一步研究Fab-HCAb结构和FIT-Ig结构之间的差异，本实施例利用已知的人IgG1全长抗体晶体结构(PDB登录号1HZH)通过同源建模技术预测出Fab-HCAb(结构(1))的三维结构模型(图17(A))和FIT-Ig(结构(5))的三维结构模型，并在此基础上测量不同抗原结合位点之间的相对距离(图17(B)和(C))；在这两个结构模型中，蛋白功能区A和蛋白功能区B之间都采用7个氨基酸长度的连接肽GS_7(SEQ ID NO：163)进行联结。
如图17中所示，Fab-HCAb结构更加紧凑。在Fab-HCAb结构处于最为伸展状态的模型中，两个VH端(B1和B2)之间距离约为10nm，两个Fab端(A1和A2)之间距离约为30nm；相应地，在FIT-Ig结构中，B1和B2之间距离约为18nm，A1和A2之间距离约为37nm。在靶向4-1BB的双特异性结合蛋白中，Fab-HCAb的这种更加紧凑的结构特性也许更有利于4-1BB的三聚化并在细胞表面成簇，进而激活下游信号。
实施例5 B7H4×4-1BB双特异性结合蛋白
在本实施例中，我们构建了靶向B7H4和4-1BB的具有Fab-HCAb、IgG-VH或VH-IgG结构的双特异性结合蛋白B7H4×4-1BB，通过一个或者多个作用机制来提高抗肿瘤效果和安全性。第一，B7H4×4-1BB可以通过解除B7H4的负调控信号来激活T细胞。第二，B7H4×4-1BB富集于B7H4高表达的肿瘤组织，在肿瘤微环境中，免疫细胞和肿 瘤细胞通过B7H4×4-1BB结合在一起，促进免疫突触的形成；同时，高表达于肿瘤细胞表面的B7H4分子可以通过B7H4×4-1BB促进T细胞表面的4-1BB分子的交联，并激活下游信号传导通路，提供共刺激信号，进而促进T细胞的活化和增殖，提高抗肿瘤活性。第三，B7H4×4-1BB只能在肿瘤微环境中利用靶细胞来介导T细胞的激活，以避免类似Urelumab的单抗在正常组织中过度激活T细胞所带来的毒副作用。
实施例5.1 获得抗B7H4的IgG抗体和抗4-1BB的HCAb抗体
实施例5.1.1 获得抗B7H4的全人源IgG抗体
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人B7H4-mFc融合蛋白(Sino Biological Inc.，#10738-H05H)对Harbour H2L2小鼠进行多轮免疫。当检测小鼠血清中B7H4特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，鉴定出若干个特异识别B7H4的单克隆抗体分子。对这些单克隆抗体进行进一步的鉴定，根据其对人B7H4的结合能力、食蟹猴B7H4的结合能力、靶细胞受体内化能力等参数，优选出数个候选抗体分子。然后对候选抗体分子进行序列分析和优化，得到数个变体序列。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。
抗B7H4的重组全人源IgG抗体PR002408的序列见表6。
实施例5.1.2 获得抗4-1BB的全人源HCAb抗体
本实施例使用的抗4-1BB的全人源HCAb抗体PR001760(表6)来源于Harbour HCAb小鼠，其发现过程如实施例4.1.3所述。
实施例5.2 构建B7H4×4-1BB双特异性结合蛋白
一方面，本实施例利用抗B7H4的IgG抗体PR002408的Fab，和抗4-1BB的HCAb抗体PR001760的VH，来构建具有如实施例1.1.1所述Fab-HCAb结构的抗B7H4×4-1BB的双特异性结合蛋白PR004279。PR004279的分子设计见表4，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表9。
另一方面，本实施例还利用抗B7H4的IgG抗体PR002408的Fab，和抗4-1BB的HCAb抗体PR001760的VH，来构建具有如实施例1.1.3所述IgG-VH结构的抗B7H4×4-1BB的双特异性结合蛋白PR003335。PR003335的分子设计见表5，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表10。
另一方面，本实施例还利用抗B7H4的IgG抗体PR002408的Fab，和抗4-1BB的HCAb抗体PR001760的VH，来构建具有如实施例1.1.4所述VH-IgG结构的抗B7H4×4-1BB的双特异性结合蛋白PR004278。PR004278的分子设计见表5，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表10。
实施例5.3 结合4-1BB
本实施例利用实施例4.3所述方法测试结合蛋白与高表达人4-1BB的CHO-K1细胞株CHO-K1/hu4-1BB(南京金斯瑞，M00538)细胞的结合能力。
图5中所示，B7H4×4-1BB双特异性结合蛋白(PR004279，PR004278，PR003335)都可以结合4-1BB；而且Fab-HCAb结构的分子(PR004279)和VH-IgG结构的分子(PR004278)结合4-1BB的能力优于IgG-VH结构的分子(PR003335)。
实施例5.4 结合B7H4
本实施例利用流式细胞术FACS测试结合蛋白与高表达人B7H4的肿瘤细胞系SK-BR-3(ATCC，HTB-30)的结合能力。具体地，消化SK-BR-3细胞并用完全培养基重悬，将细胞密度调整为2x10 6细胞/mL；接着以50μL细胞/孔接种于96孔V底板(Corning，#3894)。随后以50μL/孔加入5倍浓度梯度稀释的结合蛋白共8个浓度，混合均匀；hIgG1 iso(CrownBio，#C0001)作为同型对照。将细胞放置于4℃，避光孵育2小时。随后以100μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。之后，以100μL/孔加入荧光二抗(Alexa Fluor 647-conjugated AffiniPure Goat Anti-Human IgG，FcγFragment Specific，Jackson ImmunoResearch，#109-605-098，1∶1000稀释)，放置于4℃避光孵育1小时。随后以100μL/孔加入预冷的PBS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合蛋白对靶细胞的结合曲线及EC50值等参数。
图6中所示，B7H4×4-1BB双特异性结合蛋白(PR004279，PR004278，PR003335)都可以结合B7H4；而且Fab-HCAb结构的分子(PR004279)结合B7H4的能力略优于其他结构的分子。
实施例5.5 靶细胞介导的T细胞的特异性激活
本实施例是为了研究B7H4×4-1BB双特异结合蛋白在靶细胞的存在时通过结合4-1BB来激活T细胞的活性。靶细胞可以是高表达人B7H4的细胞SK-BR-3(ATCC，HTB-30)；效应细胞可以是分离的人PBMC或者T细胞。
具体的，首先将抗CD3抗体OKT3(Thermo，#16-0037-81)包板于96孔板(Corning，#3799)。接着，将人T细胞的密度调整为3x10 6细胞/mL，将靶细胞的密度调整为3x10 5细胞/mL，随后把两种细胞悬液各以50μL/孔接种于96孔板，最终效靶比为10∶1。然后，以50μL/孔加入5倍浓度梯度稀释的结合蛋白共5个浓度，最大终浓度为6nM，两个复孔加样；30nM的hIgG1 iso(CrownBio，#C0001)作为对照。将96孔板置于37℃，5％CO 2培养箱中孵育。分别收集培养48小时后和72小时后的上清液，用IL-2 ELISA试剂盒(Thermo，#88-7025-88)检测48小时后的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo，#88-7316-77)检测72小时后的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。应用软件GraphPad Prism 8进行数据处理和作图分析。
本实施例中，阳性对照分子为抗4-1BB的单抗Urelumab。
图7显示了结合蛋白激活T细胞释放IL-2。Fab-HCAb结构的分子(PR004279)和IgG-VH结构的分子(PR003335)有比Urelumab更强的激活T细胞的能力，且PR004279比PR003335略强。尽管VH-IgG结构的分子(PR004278)有很强的结合4-1BB的能力，但是它几乎不能激活T细胞。这说明，当4-1BB结合域VH位于IgG重链的N端时，其靶细胞结合结构域Fab和4-1BB结合域VH之间的距离不适合形成靶细胞和T细胞之间的相互作用。总体说来，T细胞激活能力排序：PR004279＞PR003335＞Urelumab＞PR004278。
实施例6 B7H4×OX40双特异性结合蛋白
在本实施例中，我们构建了靶向B7H4和OX40的具有Fab-HCAb或IgG-VH结构的双特异性结合蛋白B7H4×OX40，利用与B7H4×4-1BB类似的作用机制，通过肿瘤相关抗原B7H4将OX40抗体重定向到肿瘤细胞，特异性激活肿瘤微环境的免疫反应。
实施例6.1 获得抗B7H4的IgG抗体和抗OX40的HCAb抗体
实施例6.1.1 获得抗B7H4的全人源IgG抗体
本实施例使用的抗B7H4的重组全人源IgG抗体PR002408(表6)来源于Harbour H2L2小鼠，其发现过程如实施例5.1.1所述。
实施例6.1.2 获得抗OX40的全人源HCAb抗体
本实施例使用的抗OX40的全人源HCAb抗体PR002067(表6)来源于Harbour HCAb小鼠，其发现过程与实施例4.1.3所述的抗4-1BB的HCAb的发现过程类似，即利用重组人OX40-Fc融合蛋白(睿智化学提供)或者高表达人OX40的细胞株HEK293/OX40(睿智化学提供)对HarbourHCAb小鼠进行多轮免疫，并经过多轮筛选验证得到。
实施例6.2 构建B7H4×OX40双特异性结合蛋白
一方面，本实施例利用抗B7H4的IgG抗体PR002408的Fab，和抗OX40的HCAb抗体PR002067的VH，来构建具有如实施例1.1.1所述Fab-HCAb结构的抗B7H4×OX40的双特异性结合蛋白PR004277。PR004277的分子设计见表4，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表9。
另一方面，本实施例还利用抗B7H4的IgG抗体PR002408的Fab，和抗OX40的HCAb抗体PR002067的VH，来构建具有如实施例1.1.3所述IgG-VH结构的抗B7H4×OX40的双特异性结合蛋白PR004276。PR004276的分子设计见表5，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表10。
实施例6.3 结合OX40
本实施例利用流式细胞术FACS测试结合蛋白与高表达人OX40的CHO-K1细胞株CHO-K1/huOX40(南京金斯瑞，M00561)细胞的结合能力。具体地，消化细胞并用F12K完全培养基重悬，将细胞密度分别调整为1×10 6细胞/ml。以100μL细胞/孔接种于96孔V底板(Corning，#3894)，随后加入100μl/孔，2倍于终浓度的3倍浓度梯度稀释的待测结合蛋白。将细胞放置于4℃，避光孵育1小时。之后，加入100μl/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μl/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG，FcγFragment Specific，Jackson ImmunoResearch，#109-545-06，1∶1000稀释)，4℃，避光孵育30分钟。用100μl/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μl/孔预冷PBS重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合蛋白对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗OX40的单抗Pogalizumab(蛋白编号PR003475)。
图9中所示，B7H4×OX40双特异性结合蛋白(PR004277，PR004276)都可以结合 OX40，结合能力相当。
实施例6.4 结合B7H4
本实施例利用实施例5.4所述方法测试结合蛋白与高表达人B7H4的肿瘤细胞系SK-BR-3(ATCC，HTB-30)的结合能力。阳性对照分子为抗B7H4的单抗PR002408，亦为B7H4×OX40的B7H4端的亲本单抗。
图10中所示，B7H4×OX40双特异性结合蛋白(PR004277，PR004276)都可以结合B7H4，且与亲本抗体PR002408的结合能力一致。
实施例6.5 靶细胞介导的T细胞的特异性激活
本实施例是为了研究B7H4×OX40双特异结合蛋白在靶细胞的存在时通过结合OX40来激活T细胞的活性。靶细胞可以是高表达人B7H4的细胞CHO-K1/hB7H4(和铂医药自制)；效应细胞可以是分离的人PBMC或者T细胞。
具体的，首先以100μL/孔将0.3μg/mL抗CD3抗体OKT3(Thermo，#16-0037-81)包板于96孔板(Corning，#3599)。接着，将人T细胞(从人PBMC中用T细胞分选试剂盒(Miltenyi，#130-096-535)分离得到)的密度调整为2x10 6细胞/mL，将靶细胞的密度调整为3x10 5细胞/mL，随后把两种细胞悬液各以50μL/孔接种于96孔板。然后，以100μL/孔加入不同浓度的结合蛋白，两个复孔加样，结合蛋白终浓度为(20nM，2nM，0nM)；hIgG1 iso(CrownBio，#C0001)和不加抗体的空白孔(no Ab)作为对照。将96孔板置于37℃，5％CO 2培养箱中孵育3天。分别收集培养48小时后和72小时后的上清液，用IL-2ELISA试剂盒(Thermo，#88-7025-88)检测48小时后的上清中IL-2浓度，用IFN-γELISA试剂盒(Thermo，#88-7316-77)检测72小时后的上清中IFN-γ浓度。ELISA检测方法参照相关试剂盒操作说明。应用软件GraphPad Prism 8进行数据处理和作图分析。
本实施例中，对照分子为相应的亲本单抗PR002408和PR002067。
图11中所示，在高表达B7H4的CHOK1/hB7H4细胞存在时，抗OX40的HCAb单抗PR002067和抗B7H4的IgG单抗PR002408都不能激活T细胞；B7H4×OX40双特异性结合蛋白(PR004277，PR004276)都可以激活T细胞并促进细胞因子IL-2的产生，这说明B7H4×OX40对T细胞的激活是依赖于靶细胞的。而且，Fab-HCAb结构的分子(PR004277)的T细胞激活能力略强于IgG-VH结构的分子(PR004276)。
实施例7 BCMA×BCMA双特异性结合蛋白
在本实施例中，我们构建了靶向BCMA的具有Fab-HCAb结构的多价双表位双特异 性结合蛋白，其可以更好地利用内化作用实现对靶细胞的杀伤。
实施例7.1 获得抗BCMA的抗体
实施例7.1.1 获得抗BCMA的全人源IgG抗体
本实施例使用的抗BCMA的重组全人源IgG抗体PR000892(序列见表6)来源于Harbour H2L2小鼠，其发现过程和序列公开于发明专利CN111234020B。
实施例7.1.2 获得抗BCMA的全人源HCAb抗体
用可溶的重组人BCMA-ECD-Fc融合蛋白(ACRO Biosystems，#BC7-H82F0)对Harbour HCAb小鼠进行多轮免疫。用类似实施例4.1.3所述方法进行筛选并获得抗BCMA的全人源HCAb抗体。然后，进一步对HCAb抗体PR001046的VH的CDR区进行两轮定点突变，以获得结合BCMA的亲和力提高的突变体，如PR001046_R2_4G10(即PR004433)。
本实施例使用的抗BCMA的重组全人源HCAb抗体PR004433的序列见表6。
实施例7.2 构建BCMA×BCMA双特异性结合蛋白
本实施例利用抗BCMA的IgG抗体PR000892的Fab，和抗BCMA的HCAb抗体PR004433的VH，来构建具有如实施例1.1.1所述Fab-HCAb结构的抗BCMA×BCMA的双特异性结合蛋白PR005744。PR005744的分子设计见表4，相应的序列编号见表7；该分子按照实施例2所述方法进行制备和分析，总结于表9。
随后，测试抗原结合蛋白PR005744的结合BCMA的能力和其在BCMA高表达细胞NCI-H929(ATCC，CRL-9068)上内化的能力。
实施例7.3 利用BLI方法测定BCMA结合蛋白与BCMA的亲和力
本实施例通过生物膜干涉(BLI)技术，使用Octet分子相互作用分析仪(ForteBio，型号Octet Red96e)，来进行BCMA结合蛋白与BCMA的结合动力学分析。
将重组人BCMA-ECD-Fc融合蛋白(ACRO Biosystems，#BC7-H82F0)先利用生物素化试剂盒(EZ-Link Sulfo-NHS-LC-Biotin，ThermoFisher，A39257)按照说明书要求进行生物素化。本实验使用传感器为SA生物传感器(ForteBio，#18-5019)；工作缓冲液为1×动力学缓冲液(从10×动力学缓冲液(ForteBio，#18-1105)稀释)，用于亲和力测试以及抗原和结合蛋白的稀释；平衡缓冲液为1×PBS缓冲液(从10×PBS缓冲液(BBI Life Sciences，#E607016-0500)稀释)。
先将两列SA传感器(每列放置8个传感器；第一列称为参照SA传感器，第二列称为测试SA传感器)在平衡缓冲液中平衡10分钟。然后，测试SA传感器捕获生物素化 的BCMA，设置捕获高度为0.2nm，而参照SA传感器浸入缓冲液中30秒。两列传感器再与梯度稀释的待测BCMA结合蛋白进行结合；待测BCMA结合蛋白浓度为10-2.5nM的两倍梯度稀释以及0nM。传感器与待测蛋白结合180秒，然后解离800秒。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择双扣除模式(double reference)扣除参照信号，选择“1∶1 Global fitting”方法进行数据拟合，计算出抗原与抗原结合蛋白结合的动力学参数，得到k on(1/Ms)值、k dis(1/s)值和K D(M)值。
结果如表13和图13所示，四价的PR005744比二价的PR004433有更高的结合BCMA的亲和力(KD值)；而且PR005744比PR004433有更高的最大反应信号(Response)。
表13结合蛋白与BCMA的结合动力学参数
Figure PCTCN2021102935-appb-000025
实施例7.4 用FACS方法测定结合蛋白的内化作用
前述实施例已经验证了四价结合蛋白(PR005744)与二价结合蛋白(PR004433)相比有相似甚至更高的结合BCMA的结合能力。本实施例进一步利用FACS方法来研究靶向BCMA的抗原结合蛋白内化介导对表达人BCMA的细胞的杀伤。具体地，将NCI-H929(ATCC，CRL-9068)细胞以2×10 5个/孔接种至96-孔板(Beyotime，#FT018)；随后加入用FACS缓冲液稀释的200nM待测抗原结合蛋白；然后置于4℃孵育1小时；接着，取样品于37℃分别孵育不同时间(如30分钟、1小时、2小时和4小时)；然后，离心并重悬细胞，加入荧光二抗(Jackson ImmunoResearch，#109-545-098)后再于4℃孵育30分钟。最后，使用流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。以37℃孵育0分钟(T0)时的荧光信号MFI为基线，不同孵育时间的样品的MFI与T0的基线进行扣减并计算相对减少量，以此反映抗原结合蛋白的内化作用的效率。应用软件GraphPad Prism 8进行数据处理和作图分析。
如图12所示，PR005744在NCI-H929细胞的内化作用显著优于PR004433；其在30分钟内可以使超过60％的BCMA被内化。
实施例8 HER2×CTLA4双特异性结合蛋白
在本实施例中，我们构建了多个靶向HER2和CTLA4的具有Fab-HCAb结构的双特异性结合蛋白HER2×CTLA4。HER2×CTLA4可以富集于HER2高表达的肿瘤组织，在肿瘤微环境中特异性地解除CTLA4抑制信号来激活T细胞，减少CTLA4单抗在外周系统非特异激活带来的毒副作用。本实施例构建了多个含有不同连接肽的Fab-HCAb结构的分子，以研究连接肽对Fab-HCAb分子结构的影响。
实施例8.1 获得抗HER2的IgG抗体和抗CTLA4的HCAb抗体
实施例8.1.1 获得抗HER2的IgG抗体
本实施例使用抗HER2的IgG抗体trastuzumab(蛋白编号PR000210)，其相应的氨基酸序列来源于IMGT数据库，序列见表6。
实施例8.1.2 获得抗CTLA4的全人源HCAb抗体
用可溶的重组人CTLA4蛋白(ACRO Biosystems，#CT4-H5229)对Harbour HCAb小鼠进行多轮免疫。用类似实施例4.1.3所述方法进行筛选并获得抗CTLA4的全人源HCAb抗体。
本实施例使用的抗CTLA4的重组全人源HCAb抗体PR000184的序列见表6。
实施例8.2 构建HER2×CTLA4双特异性结合蛋白
本实施例利用抗HER2的IgG抗体PR000210(trastuzumab类似物)的Fab，和抗CTLA4的HCAb抗体PR000184的VH，来构建具有如实施例1.1.1所述Fab-HCAb结构的抗HER2×CTLA4的双特异性结合蛋白PR000305、PR000653、PR000654、PR000655和PR000706。其分子设计见表4，相应的序列编号见表7；并按照实施例2所述方法进行制备和分析，总结于表9。这些双特异性结合蛋白分子具有相似的结构，其抗原结合结构域Fab和VH都相同，其细微的差异在于不同的第一连接肽(Fab和VH之间)和第二连接肽(VH和CH2之间)的序列。
本实施例利用这些分子来研究不同连接肽对Fab-HCAb分子结构的影响。
实施例8.3 结合HER2
本实施例利用流式细胞术FACS测试结合蛋白与高表达人HER2的肿瘤细胞系SK-BR-3(ATCC，HTB-30)的结合能力。具体地，消化SK-BR-3细胞，并用完全培养基重悬， 将细胞密度调整为1x10 6细胞/mL；接着以100μL细胞/孔接种于96孔V底板(Corning，#3894)，4℃下离心5分钟，弃上清。随后以100μL/孔加入以最高终浓度为100nM的5倍浓度梯度稀释的结合蛋白，共8个浓度，混合均匀；hIgG1 iso(CrownBio，#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。之后，4℃下离心5分钟，弃上清；随后以200μL/孔加入预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，然后于500g，4℃下离心5分钟，弃上清。之后，以100μL/孔加入荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction，Thermo，#A11013，1∶1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗HER2的单抗PR000210(trastuzumab类似物)，亦为HER2×CTLA4的HER2端的亲本单抗。
图14中所示，Fab-HCAb结构的双特异性结合蛋白(PR000305、PR000653、PR000654、PR000655和PR000706)结合HER2的能力与亲本单抗PR000210相当，体现在几乎一致的EC50值和MFI最大值。这说明，Fab-HCAb结构的Fab端可以很好地保留其对应的靶点结合能力。
实施例8.4 结合CTLA4
本实施例利用流式细胞术FACS测试结合蛋白与高表达人CTLA4的CHO-K1细胞株CHO-K1/hCTLA4(睿智化学)等细胞的结合能力。具体地，消化CHO-K1/hCTLA4细胞，并用F12K培养基重悬；将细胞密度调整为2x10 6细胞/mL。接着将CHO-K1/hCTLA4细胞以100μL/孔接种于96孔V底板(Corning，#3894)，4℃下离心5分钟，弃上清。随后以100μL/孔加入以最高终浓度为300nM的5倍浓度梯度稀释的结合蛋白，共8个浓度，混合均匀；hIgG1 iso(CrownBio，#C0001)作为同型对照。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Goat human lgG(H+L)Alexa Fluor 488 conjunction，Thermo，#A11013，1∶1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬 细胞。使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo，LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
本实施例中，阳性对照分子为抗CTLA4的HCAb单抗PR000184，亦为HER2×CTLA4的CTLA4端的亲本单抗。
图15中所示，Fab-HCAb结构的双特异性结合蛋白(PR000305、PR000653、PR000654、PR000655和PR000706)都可以结合CTLA4。这些分子具有相似的结构，CTLA4端的VH序列也相同，其细微的差异在于不同的第一连接肽和连接Fc的铰链区；因而这些分子有非常相似的结合CTLA4的能力。这说明了不同的连接肽的长度或者序列对Fab-HCAb结构中的结合结构域VH的影响很小。
另一方面，这些分子结合CTLA4的EC50值与亲本单抗PR000184的相似或者略弱仅1.5～3倍，但是在FACS上的最大结合信号(MFI最大值)比亲本单抗PR000184低。这可能暗示了，在Fab-HCAb结构的某些应用场景中，Fab结构域可能对HCAb的VH结构域有一种“遮蔽”效应，使得Fab-HCAb分子可能会优先结合Fab结构域识别的靶点，之后才会引起VH结构域的结合。这种先后顺序的结合以及不同靶点结合力的差异可以适用于一些特殊的应用场景的需求。例如，抗HER2的单抗trastuzumab在治疗乳腺癌的推荐初始剂量是4mg/kg，在治疗胃癌的推荐初始剂量是8mg/kg；而抗CTLA4的单抗ipilimumab在治疗黑色素瘤的推荐剂量3mg/kg，在组合疗法中剂量更低。Fab-HCAb结构的HER2×CTLA4的HER2端的活性几乎与其亲本单抗相当，但是其CTLA4端的活性相对减弱，因而可以用来实现临床上对CTLA4抑制剂的中低剂量的需求。此外，HER2×CTLA4可以优先结合HER2，使其富集于HER2高表达的肿瘤组织，进而减少CTLA4抗体在外周系统非特异激活T细胞带来的毒副作用。
实施例9 Fab-HCAb结构的分子的药代动力学研究
本实施例研究了具有Fab-HCAb结构的双特异性结合蛋白分子PR004270(序列见表7)在小鼠体内的药代动力学性能。
给药和采血：对于每一个测试抗体分子，选取体重18～22克的雌性BALB/c或者C57BL/6小鼠6只，按5mg/kg的剂量通过静脉注射给与测试抗体分子。一组3只于给药前以及给药后15分钟、24小时(1天)、第4天、和第10天采集全血，另一组3只于只于给药前以及给药后5小时、第2天、第7天、和第14天采集全血。将全血静置30 分钟使其凝固，随后离心并将分离的血清样品在-80℃下冻存直至分析。
分析方法：采用两种ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法一，即Fc端检测方法，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的抗体，然后加入HRP标记的山羊抗人Fc第二抗体来检测。ELISA方法二，即功能结构域检测方法，通过包被于96孔板的PD-L1蛋白来捕获小鼠血清中的特异识别抗原的抗体，然后加入HRP标记的的山羊抗人Fc第二抗体来检测。最后，使用Phoenix WinNonlin软件8.2版，选用非房室模型(NCA)分析药代动力学参数。
图16和表14中所示，Fab-HCAb结构的分子PR004270有与常规IgG抗体相似的血清半衰期t 1/2值，PD-L1端检测方法显示其t 1/2值超过10天。
表14小鼠体内的药代动力学参数
Figure PCTCN2021102935-appb-000026
本实施例说明了Fab-HCAb结构的分子具有优秀的药代动力学性能。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (21)

  1. 一种含有至少两个蛋白功能区的结合蛋白，其中，所述结合蛋白包括蛋白功能区A和蛋白功能区B；所述蛋白功能区A和所述蛋白功能区B靶向不同的抗原或相同抗原的不同表位；
    其中所述蛋白功能区A为Fab结构；所述蛋白功能区B为VH结构，所述结合蛋白还包括Fc同源二聚体；
    所述蛋白功能区A的数量为二个，所述蛋白功能区B的数量为二个；所述结合蛋白为左右对称的结构；
    所述结合蛋白从N末端至C末端依次为蛋白功能区A、蛋白功能区B和Fc同源二聚体，其中所述蛋白功能区A与所述蛋白功能区B通过第一连接肽(L1)连接，所述蛋白功能区B与所述Fc通过第二连接肽(L2)连接。
  2. 如权利要求1所述的结合蛋白，其中，所述结合蛋白具有四条多肽链，分别为两条相同的短链和两条相同的长链，其中，(1)所述短链从N末端至C末端依次包括VH_A-CH1，所述长链从N末端至C末端依次包括VL_A-CL-L1-VH_B-L2-CH2-CH3；或(2)所述短链从N末端至C末端依次包括VL_A-CL，所述长链从N末端至C末端依次包括VH_A-CH1-L1-VH_B-L2-CH2-CH3；
    其中，所述的VL_A和VH_A分别为所述蛋白功能区A的VL和VH，所述的VH_B为所述蛋白功能区B的VH；所述的CL是轻链恒定区结构域；所述的CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域；所述的L1或L2为连接肽；
    较佳地，所述L1和L2独立地为例如“-”、GS或如SEQ ID NOs：161-182的氨基酸序列所示。
  3. 如权利要求1或2所述的结合蛋白，其中，所述抗原选自PD-L1、HER2、B7H4、CTLA4、OX40、4-1BB和BCMA中的一种或多种；
    较佳地，
    所述蛋白功能区A为来源于PD-L1抗体或其抗原结合片段、HER2抗体或其抗原结合片段、B7H4抗体或其抗原结合片段或BCMA抗体或其抗原结合片段的Fab，和/或，所述蛋白功能区B为来源于CTLA4抗体或其抗原结合片段、4-1BB抗体或其抗原结合片段、OX40抗体或其抗原结合片段或BCMA抗体或其抗原结合片段的VH；
    更佳地，
    所述蛋白功能区A为来源于HER2抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于CTLA4抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于 PD-L1抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于4-1BB抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于B7H4抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于4-1BB抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于B7H4抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于OX40抗体或其抗原结合片段的VH；或，所述蛋白功能区A为来源于BCMA抗体或其抗原结合片段的Fab，且所述蛋白功能区B为来源于BCMA抗体或其抗原结合片段的VH。
  4. 如权利要求3所述的结合蛋白，其中，
    所述的PD-L1抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：75、85和97所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、32和54所示；
    所述的B7H4抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：78、83和100所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：15、37和59所示；
    所述的4-1BB抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：73、83和95所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：11、30和52所示；或者，所述的4-1BB抗体或其抗原结合片段包含重链可变区(VH)；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：14、35和57所示；
    所述的OX40抗体或其抗原结合片段包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、36和58所示；
    所述的BCMA抗体或其抗原结合片段包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：17、39和61所示；或者，所述的BCMA抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：77、87和99所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、34和56所示；
    所述的CTLA4抗体或其抗原结合片段包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：10、29和51所示；和/或
    所述的HER2抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)， 所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：74、84和96所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：12、31和53所示。
  5. 如权利要求3或4所述的结合蛋白，其中，
    所述的PD-L1抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：118所示的氨基酸序列，所述VH包括如SEQ ID NO：108所示的氨基酸序列；
    所述的B7H4抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：121所示的氨基酸序列，所述VH包括如SEQ ID NO：113所示的氨基酸序列；
    所述的4-1BB抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：116所示的氨基酸序列，所述VH包括如SEQ ID NO：106所示的氨基酸序列；或者所述的4-1BB抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：111所示的氨基酸序列；
    所述的OX40抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：112所示的氨基酸序列；
    所述的BCMA抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：115所示的氨基酸序列；或者，所述的BCMA抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包括如SEQ ID NO：120所示的氨基酸序列，所述VH包括如SEQ ID NO：110所示的氨基酸序列；
    所述的CTLA4抗体或其抗原结合片段包含重链可变区(VH)，所述VH包括如SEQ ID NO：105所示的氨基酸序列；和/或
    所述的HER2抗体或其抗原结合片段包含轻链可变区(VL)和重链可变区(VH)，所述VL包含如SEQ ID NO：117所示的氨基酸序列，所述VH包含如SEQ ID NO：107所示的氨基酸序列。
  6. 如权利要求1-5任一项所述的结合蛋白，其中，所述结合蛋白含有蛋白功能区A和蛋白功能区B：
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：75、85和97所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、32和54所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：14、35和57所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：78、83和100所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：15、37和59所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：14、35和57所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：78、83和100所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：15、37和59所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、36和58所示；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：77、87和99所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：13、34和56所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，所示氨基酸序列分别如SEQ ID NOs：17、39和61；或，
    所述蛋白功能区A包含轻链可变区(VL)和重链可变区(VH)，所述VL包含LCDR1、LCDR2和LCDR3，氨基酸序列分别如SEQ ID NOs：74、84和96所示；所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：12、31和53所示；并且，所述蛋白功能区B包含重链可变区(VH)，所述VH包含HCDR1、HCDR2和HCDR3，氨基酸序列分别如SEQ ID NOs：10、29和51所示。
  7. 如权利要求1-6任一项所述的结合蛋白，其中，所述结合蛋白含有蛋白功能区A和蛋白功能区B：
    所述蛋白功能区A包含氨基酸序列如SEQ ID NO：118所示的轻链可变区和氨基酸序列如SEQ ID NO：108所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：111所示的重链可变区；或，
    所述蛋白功能区A包含氨基酸序列如SEQ ID NO：121所示的轻链可变区和氨基酸序列如SEQ ID NO：113所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：111所示的重链可变区；或，
    所述蛋白功能区A包含氨基酸序列如SEQ ID NO：121所示的轻链可变区和氨基酸序列如SEQ ID NO：113所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：112所示的重链可变区；或，
    所述蛋白功能区A包含氨基酸序列如SEQ ID NO：120所示的轻链可变区和氨基酸 序列如SEQ ID NO：110所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：115所示的重链可变区；或，
    所述蛋白功能区A包含氨基酸序列如SEQ ID NO：117所示的轻链可变区和氨基酸序列如SEQ ID NO：107所示的重链可变区；所述蛋白功能区B包含氨基酸序列如SEQ ID NO：105所示的重链可变区。
  8. 如权利要求1-7任一项所述的结合蛋白，其中，所述结合蛋白包含两种不同的多肽链：第一多肽链和第二多肽链，
    所述第一多肽链包括如SEQ ID NO：147所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：153所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：136所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：183所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：147所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：184所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：155所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：158所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：155所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：156所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：159所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：160所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：141所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：142所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：141所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：143所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：141所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：144所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：141所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：145所示的氨基酸序列；或，
    所述第一多肽链包括如SEQ ID NO：141所示的氨基酸序列，和所述第二多肽链包括如SEQ ID NO：149所示的氨基酸序列。
  9. 权利要求1-8任一项所述的结合蛋白，其中，所述的结合蛋白中，所述Fab结构中的轻链恒定区(CL)为人轻链恒定区，优选人轻链恒定区Cκ或Cλ；
    和/或，所述的结合蛋白中，所包含的重链恒定区(CH)为人重链恒定区，优选人IgG1、 IgG2、IgG3、IgG4重链恒定区或其突变；所述突变优选自C220S、N297A、L234A、L235A、G237A和P329G中的一种或多种突变，所述突变位点使用EU编号规则。
  10. 一种分离的核酸，其编码如权利要求1-9任一项所述的结合蛋白。
  11. 一种重组表达载体，其包含如权利要求10所述的分离的核酸；较佳地，所述表达载体包含真核细胞表达载体和/或原核细胞表达载体。
  12. 一种转化体，其包含如权利要求10所述的分离的核酸或如权利要求11所述的重组表达载体；
    较佳地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
  13. 一种结合蛋白的制备方法，其包含培养如权利要求12中所述的转化体，从培养物中获得结合蛋白。
  14. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-9任一项所述的结合蛋白，以及药学上可接受的载体；
    较佳地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分。
  15. 一种试剂盒，其包括如权利要求1-9任一项所述的结合蛋白和/或如权利要求14所述的药物组合物；
    较佳地，所述试剂盒还包括(i)施用结合蛋白或药物组合物的装置；和/或(ii)使用说明。
  16. 一种套装药盒，其特征在于，所述套装药盒包括药盒一和药盒二，所述药盒一包括如权利要求1-9任一项所述的结合蛋白和/或如权利要求14所述的药物组合物，所述药盒二包括其它抗体或药物组合物。
  17. 一种给药装置，其特征在于，所述给药装置包括如权利要求1-9任一项所述的结合蛋白和/或如权利要求14所述的药物组合物；
    较佳地，所述给药装置还包括容纳或将所述合蛋白和/或所述药物组合物施用于受试者的部件，例如注射器、输液装置或植入式给药装置。
  18. 如权利要求1-9中任一项所述的结合蛋白、如权利要求14所述的药物组合物、如权利要求15所述的试剂盒、如权利要求16所述的套装药盒和/或如权利要求17所述的给药装置在制备诊断、预防和/或治疗癌症或其他疾病的药物中的应用；
    较佳地，所述的癌症选自乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤和多发性骨髓瘤中的一种或多种。
  19. 一种体外或体内检测特异性抗原的方法，其包括使用如权利要求1-9中任一项所述的结合蛋白进行检测。
  20. 如权利要求1-9中任一项所述的结合蛋白、如权利要求14所述的药物组合物、如权利要求15所述的试剂盒、如权利要求16所述的套装药盒和/或如权利要求17所述的给药装置在诊断、预防和/或治疗癌症或其他疾病中的应用；
    较佳地，所述的癌症选自乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤和多发性骨髓瘤中的一种或多种。
  21. 一种诊断、预防和/或治疗癌症或其他疾病的方法，所述方法包括向有需要的患者施用如权利要求1-9中任一项所述的结合蛋白、如权利要求14所述的药物组合物、如权利要求15所述的试剂盒、如权利要求16所述的套装药盒和/或如权利要求17所述的给药装置的步骤；
    较佳地，所述的癌症选自乳腺癌、卵巢癌、子宫内膜癌、肾癌、黑色素瘤、肺癌、胃癌、肝癌、食管癌、宫颈癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、肉瘤、结直肠癌、淋巴瘤和多发性骨髓瘤中的一种或多种。
PCT/CN2021/102935 2020-06-30 2021-06-29 Fab-HCAb结构的结合蛋白 Ceased WO2022002006A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202180040307.2A CN115667316A (zh) 2020-06-30 2021-06-29 Fab-HCAb结构的结合蛋白
US18/013,530 US20230287143A1 (en) 2020-06-30 2021-06-29 Binding protein in fab-hcab structure

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN202010630471.6 2020-06-30
CN202010618158 2020-06-30
CN202010630471 2020-06-30
CN202010618158.0 2020-06-30
CN202011423832.6 2020-12-08
CN202011423832 2020-12-08

Publications (1)

Publication Number Publication Date
WO2022002006A1 true WO2022002006A1 (zh) 2022-01-06

Family

ID=79317455

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/102935 Ceased WO2022002006A1 (zh) 2020-06-30 2021-06-29 Fab-HCAb结构的结合蛋白

Country Status (4)

Country Link
US (1) US20230287143A1 (zh)
CN (1) CN115667316A (zh)
TW (1) TWI818276B (zh)
WO (1) WO2022002006A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024131731A1 (zh) * 2022-12-19 2024-06-27 和铂医药(上海)有限责任公司 "κ/λ"Fab-Fab串联多特异性结合蛋白及其制备和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009068649A2 (en) * 2007-11-30 2009-06-04 Glaxo Group Limited Antigen-binding constructs
WO2011036460A1 (en) * 2009-09-25 2011-03-31 Ucb Pharma S.A. Disulfide stabilised multivalent antibodies
CN109476762A (zh) * 2016-07-20 2019-03-15 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180346571A1 (en) * 2015-11-17 2018-12-06 Oncomed Pharmaceuticals, Inc. Pd-l1-binding agents and uses thereof
WO2018027204A1 (en) * 2016-08-05 2018-02-08 Genentech, Inc. Multivalent and multiepitopic anitibodies having agonistic activity and methods of use

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009068649A2 (en) * 2007-11-30 2009-06-04 Glaxo Group Limited Antigen-binding constructs
WO2011036460A1 (en) * 2009-09-25 2011-03-31 Ucb Pharma S.A. Disulfide stabilised multivalent antibodies
CN109476762A (zh) * 2016-07-20 2019-03-15 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AHAMADI-FESHARAKI RAOUFEH, FATEH ABOLFAZL, VAZIRI FARZAM, SOLGI GHASEM, SIADAT SEYED DAVAR, MAHBOUDI FEREIDOUN, RAHIMI-JAMNANI FAT: "Single-Chain Variable Fragment-Based Bispecific Antibodies: Hitting Two Targets with One Sophisticated Arrow", MOLECULAR THERAPY - ONCOLYTICS, vol. 14, 1 September 2019 (2019-09-01), pages 38 - 56, XP055798141, ISSN: 2372-7705, DOI: 10.1016/j.omto.2019.02.004 *
AIFEN LI, JIEYU XING, LI LI, CHANGHUA ZHOU, BIN DONG, PING HE, QING LI, ZHONG WANG: "A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells", AMB EXPRESS, SPRINGER BERLIN HEIDELBERG, vol. 6, no. 1, 1 December 2016 (2016-12-01), XP055604153, DOI: 10.1186/s13568-016-0201-4 *
HUANG SHUYU; SEGUéS AINA; HULSIK DAVID LUTJE; ZAISS DIETMAR M.; SIJTS ALICE J.A.M.; VAN DUIJNHOVEN SANDER M.J.; VAN ELSAS AND: "A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM., NL, vol. 483, 19 June 2020 (2020-06-19), NL , XP086209405, ISSN: 0022-1759, DOI: 10.1016/j.jim.2020.112811 *
LI LI, HE PING, ZHOU CHANGHUA, JING LI, DONG BIN, CHEN SIQI, ZHANG NING, LIU YAWEI, MIAO JI, WANG ZHONG, LI QING: "A Novel Bispecific Antibody, S-Fab, Induces Potent Cancer Cell Killing", JOURNAL OF IMMUNOTHERAPY, LIPPINCOTT WILLIAMS & WILKINS, US, vol. 38, no. 9, 1 November 2015 (2015-11-01), US , pages 350 - 356, XP055319111, ISSN: 1524-9557, DOI: 10.1097/CJI.0000000000000099 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024131731A1 (zh) * 2022-12-19 2024-06-27 和铂医药(上海)有限责任公司 "κ/λ"Fab-Fab串联多特异性结合蛋白及其制备和应用

Also Published As

Publication number Publication date
CN115667316A (zh) 2023-01-31
TWI818276B (zh) 2023-10-11
US20230287143A1 (en) 2023-09-14
TW202202526A (zh) 2022-01-16

Similar Documents

Publication Publication Date Title
US20230406932A1 (en) Antibodies that modulate a biological activity expressed by a cell
AU2018218753A1 (en) Anti-GPRC5D antibody and molecule containing same
CN111094348B (zh) 双特异性抗pd1-抗tim3抗体
TWI806087B (zh) 免疫細胞銜接多特異性結合蛋白及其製備和應用
RS59480B1 (sr) Pd-1 antitelo, njegov fragment koji se vezuje na antigen, i njegova medicinska primena
TW202132351A (zh) 抗cd47/抗pd-l1抗體及其應用
TWI815136B (zh) 一種雙特異性抗體及其用途
TWI806088B (zh) 具有H2L2與HCAb結構的結合蛋白
US20240124563A1 (en) Anti-Human MSLN Antibody And Application Thereof
WO2023011654A1 (zh) 抗pd-1纳米抗体及其应用
JP2023524102A (ja) Ａｂｃｂ５に特異的な抗体およびその使用
TW202246340A (zh) 抗ctla-4抗體及其應用
US20250340631A1 (en) Cldn18.2-targeting antibody, bispecific antibody and use thereof
CN114945596A (zh) 用于调节免疫细胞衔接效应的手段和方法
TWI818276B (zh) Fab-HCAb結構的結合蛋白
WO2023186113A1 (zh) 靶向pd-l1和cd40的抗原结合蛋白及其制备和应用
WO2022247826A1 (zh) 靶向pd-l1和cd73的特异性结合蛋白
TWI833227B (zh) 靶向pd-l1和cd73的特異性結合蛋白及其應用
JP7790754B2 (ja) Ｃｄ３を標的とする抗体、二重特異性抗体及びその使用
HK40082471A (zh) 具有h2l2与hcab结构的结合蛋白
WO2022063314A1 (zh) 靶向PD-1或PD-L1和TGF-β的双功能蛋白及其医药用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21834158

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21834158

Country of ref document: EP

Kind code of ref document: A1