WO2023051680A1 - 针对免疫检查点的双特异性抗体 - Google Patents

针对免疫检查点的双特异性抗体 Download PDF

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WO2023051680A1
WO2023051680A1 PCT/CN2022/122531 CN2022122531W WO2023051680A1 WO 2023051680 A1 WO2023051680 A1 WO 2023051680A1 CN 2022122531 W CN2022122531 W CN 2022122531W WO 2023051680 A1 WO2023051680 A1 WO 2023051680A1
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amino acid
seq
acid sequence
sequence shown
variable region
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PCT/CN2022/122531
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English (en)
French (fr)
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王亮亮
张正平
袁伟华
李奕萌
张莹
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正大天晴药业集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to bispecific antibodies, in particular to bispecific antibodies targeting CD47 and PD-L1.
  • CD47 is a transmembrane glycoprotein widely expressed in multiple species and various tissues, also known as integrin-associated protein, and is a member of the immunoglobulin superfamily.
  • Inhibitory receptor signaling regulatory protein ⁇ (SIRP ⁇ ) is one of the ligands of CD47, and CD47 binds to the NH 2 terminal IgV-like domain of SIRP ⁇ .
  • SIRP ⁇ is mainly expressed in cells of myeloid origin, including macrophages, granulocytes, DC cells, mast cells and their precursors, including hematopoietic stem cells.
  • CD47 expression and/or activation has been implicated in many diseases and disorders. Different studies have shown that almost all tumor cells and tumor tissues highly express CD47.
  • CD47 which is highly expressed on the surface of tumor cells, binds to SIRP ⁇ on the surface of macrophages and releases the "don't eat me” signal, which leads to the macrophages in the tumor tissue infiltration area not only living in harmony with tumor cells, but also It can also promote the proliferation and growth of tumor cells by promoting the proliferation of blood vessels in the tumor and inhibiting the function of effector T cells.
  • CD47 Based on the particularity of the CD47 target itself, the development of drugs targeting it often revolves around how to restore the phagocytosis of tumor cells by macrophages while avoiding its damage to normal cells. At present, the severe anemia response caused by CD47 is still a major challenge for the application of drugs targeting this target.
  • Programmed death molecule 1 and its ligand are a pair of negative immune co-stimulatory molecules.
  • PD-L1 Programmed death-ligand 1, PD-L1 or PDL1
  • CD274 and B7-H1 is a ligand of programmed death molecule 1 (Programmed death, PD-1).
  • PD-L1 Programmed death-ligand 1, PD-L1 or PDL1
  • CD274 and B7-H1 is a ligand of programmed death molecule 1 (Programmed death, PD-1).
  • the combination of PD-L1 on the surface of tissue cells and PD-1 on the surface of lymphocytes can inhibit the function of lymphocytes and induce apoptosis of activated lymphocytes, thereby playing a role in autoimmune tolerance and preventing autoimmune diseases. important role.
  • a variety of tumor cells also express PD-L1 on the surface.
  • PD-L1 expressed by tumor cells can bind to PD-1 molecules on the surface of tumor-infiltrating lymphocytes, inhibit the function of lymphocytes and the release of cytokines, and induce lymphocyte apoptosis. , so as to resist the killing effect of lymphocytes, and eventually lead to immune escape of tumors.
  • Monoclonal antibody targeting programmed death ligand 1 (PD-L1) which prevents PD-L1 from binding to PD-1 and B7-1 receptors on the surface of T cells, restores the activity of T cells, thereby enhancing the immune response, and has therapeutic Potential for multiple types of tumors.
  • the present invention provides bispecific antibodies binding to CD47 and PD-L1.
  • the present invention provides a bispecific antibody comprising a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds PD-L1; wherein the first antigen-binding portion comprises:
  • a heavy chain variable region comprising:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4 or 5,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9 or 10, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14 or 15;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18, 19 or 20,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25, and
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 26, 27, 28, 30 or 32.
  • the first antigen binding moiety comprises:
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, and HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 11 HCDR3; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26;
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 12 HCDR3; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27;
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 8
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 13 HCDR3;
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28;
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 14 HCDR3; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; or
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 15 HCDR3; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30.
  • the heavy chain variable region and the light chain variable region of the first antigen binding portion are selected from any of the following: comprising:
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 33, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 34 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 35, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 36 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 37, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 38 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:39, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO:40 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 41, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 42 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 83, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 84 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 85, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 86 amino acid sequences of at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 87, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 88 Amino acid sequences of at least 80% identity.
  • the second antigen binding portion comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, comprising SEQ ID NO: HCDR2 with the amino acid sequence shown in 200, and HCDR3 with the amino acid sequence shown in SEQ ID NO: 201
  • the light chain variable region includes: LCDR1 with the amino acid sequence shown in SEQ ID NO: 202, with SEQ ID NO: 203 LCDR2 of the amino acid sequence shown, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:204.
  • the heavy chain variable region of the second antigen-binding portion comprises an amino acid sequence at least 85% identical to the sequence set forth in SEQ ID NO: 205, and the light chain of the second antigen-binding portion can be The variable region comprises an amino acid sequence at least 80% identical to the sequence shown in SEQ ID NO: 206. In some specific embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 205, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 206.
  • the bispecific antibody comprises a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds PD-L1; wherein the first antigen-binding portion comprises: a heavy chain variable region, which Comprising: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 15; and a light chain variable region, which Comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30; the second antigen-binding portion comprises A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, HCDR2 comprising the amino acid sequence shown in S
  • the bispecific antibody comprises a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds PD-L1; wherein the first antigen-binding portion comprises: a heavy chain variable region, which Comprising an amino acid sequence shown in SEQ ID NO: 79; and a light chain variable region comprising an amino acid sequence shown in SEQ ID NO: 80; the second antigen binding portion comprising: a heavy chain variable region comprising a SEQ ID NO : the amino acid sequence shown in 205; and the light chain variable region, which comprises the amino acid sequence shown in SEQ ID NO: 206.
  • the first antigen-binding portion comprises: a heavy chain variable region, which Comprising an amino acid sequence shown in SEQ ID NO: 79; and a light chain variable region comprising an amino acid sequence shown in SEQ ID NO: 80
  • the second antigen binding portion comprising: a heavy chain variable region comprising a SEQ ID NO : the amino acid sequence shown in 205
  • the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.
  • the first antigen binding moiety and the second antigen binding moiety are each independently a Fab, ScFv, ScFab (single chain Fab), or other structures.
  • the first antigen binding moiety and the second antigen binding moiety are both Fabs.
  • one of said first antigen binding moiety and second antigen binding moiety has a domain substitution in which a heavy chain variable region and a light chain variable region are substituted for each other, or/ and the constant structure regions CH1 and CL replace each other.
  • the first antigen-binding moiety is Fab and the first antigen-binding moiety is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the Fc polypeptides of the Fc domain
  • the second The antigen binding portion is a Fab and the second antigen binding portion is fused at the C-terminus of its Fab heavy chain to the N-terminus of another Fc polypeptide of the Fc domain.
  • the bispecific antibody comprises a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds PD-L1; wherein the first antigen-binding portion is a Fab and comprises: a heavy chain A variable region comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:10, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:15; and a light chain A variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30; said No.
  • the second antigen-binding part is Fab and includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, including amino acids shown in SEQ ID NO: 200
  • the HCDR2 of sequence and comprise the HCDR3 of amino acid sequence shown in SEQ ID NO:201;
  • Light chain variable region comprises: comprise the LCDR1 of amino acid sequence shown in SEQ ID NO:202, comprise the LCDR2 of amino acid sequence shown in SEQ ID NO:203 , and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 204.
  • one of the first antigen binding moiety and the second antigen binding moiety has a domain substitution in which the constant domains CH1 and CL replace each other such that the second antigen binding moiety
  • the second antigen-binding portion comprises a peptide chain composed of a heavy chain variable region and a light chain constant region, and a peptide chain composed of a light chain variable region and a heavy chain constant region.
  • the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.
  • the Fc domain comprises an amino acid substitution that promotes association of the two Fc polypeptides of the Fc domain, or/and the Fc domain comprises an amino acid substitution that reduces binding of the Fc domain to an Fc receptor Amino acid substitutions for affinity and/or effector functions, or/and the Fc domain comprises amino acid substitutions that reduce or eliminate the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.
  • the bispecific antibody comprises a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds PD-L1; wherein the first antigen-binding portion is a Fab and comprises: a heavy chain variable A region comprising the amino acid sequence shown in SEQ ID NO: 79; and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 80; the second antigen-binding portion is a Fab and comprising: a heavy chain variable region , which comprises the amino acid sequence shown in SEQ ID NO: 205; and the light chain variable region, which comprises the amino acid sequence shown in SEQ ID NO: 206.
  • one of the first antigen binding moiety and the second antigen binding moiety has a domain substitution in which the constant domains CH1 and CL replace each other such that the second antigen binding moiety
  • the second antigen-binding portion comprises a peptide chain composed of a heavy chain variable region and a light chain constant region, and a peptide chain composed of a light chain variable region and a heavy chain constant region.
  • the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.
  • the Fc domain comprises an amino acid substitution that promotes association of the two Fc polypeptides of the Fc domain, or/and the Fc domain comprises an amino acid substitution that reduces binding of the Fc domain to an Fc receptor Amino acid substitutions for affinity and/or effector functions, or/and the Fc domain comprises amino acid substitutions that reduce or eliminate the binding of the CH3 region of an Fc polypeptide in the Fc domain to protein A.
  • the bispecific antibody consists of the following four polypeptide chains:
  • a first heavy chain comprising the heavy chain variable region of said first antigen binding portion
  • a first light chain comprising the light chain variable region of said first antigen binding portion
  • a second light chain comprising the light chain variable region of said second antigen binding portion.
  • the bispecific antibody is bivalent.
  • the invention provides an isolated nucleic acid encoding the bispecific antibody of the invention.
  • the present invention provides a vector comprising the nucleic acid of the present invention.
  • the present invention provides a host cell comprising the nucleic acid of the present invention, or the vector of the present invention.
  • the present invention provides a method for preparing the bispecific antibody, which includes the host cell to express the bispecific antibody, and separates and purifies the bispecific antibody in the system Antibody.
  • the present invention provides a pharmaceutical composition, which comprises the bispecific antibody described in the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a method for treating cancer in a subject, which comprises administering a therapeutically effective amount of the bispecific antibody or the pharmaceutical composition to the subject.
  • Figure 1 is a schematic diagram of the structure of the anti-PDL1*CD47 bispecific antibody CRS147-3355b.
  • Figure 2 is a schematic diagram of the structure of the control bispecific antibody BM.
  • Figure 3 shows the purity of the bispecific antibody CRS147-3355b detected by size exclusion chromatography (SEC).
  • Figure 4 shows the binding of anti-PDL1*CD47 bispecific antibody CRS147-3355b to CCRF-CEM-PDL1-20 cells.
  • Figure 5 shows the binding of anti-PDL1*CD47 bispecific antibody CRS147-3355b to Raji-PDL1 cells.
  • Figure 6A shows the production of IL-2 under different concentrations of antibodies in MLR
  • Figure 6B shows the production of IFN ⁇ under different concentrations of antibodies in MLR.
  • Figure 7 shows the agglutination effect of anti-PDL1*CD47 bispecific antibody CRS147-3355b on red blood cells.
  • antibody refers to a protein comprising an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, Trispecific antibody, etc.), single-chain antibody, etc.
  • multispecific means that an antibody is capable of specifically binding a plurality of different antigenic determinants, eg, capable of specifically binding two or more different antigenic determinants.
  • bispecific antibodies comprise two antigen-binding sites, each of which is specific for a different antigenic determinant.
  • Different epitopes can be expressed on the same or different cells.
  • Different antigenic determinants can be different due to different types of antigens (for example, binding antigens CD47 and PDL1), and can also exist on the same antigen.
  • An antigenic determinant is a special chemical group with a certain composition and structure on the surface or other parts of an antigenic substance molecule, which can specifically bind to its corresponding antibody or sensitized lymphocyte.
  • An example of an antigenic determinant is CD47 as an antigenic substance, which has a variety of antigenic determinants with confirmed or undetermined structures.
  • bispecific antibodies as long as an antibody that can bind to two different antigenic determinants on the antigen is considered called bispecific antibodies.
  • a particular bispecific antibody may for example bind PDL1 and CD47.
  • N-valent antibody is intended to describe the presence of N antigen-binding sites in an antibody.
  • bivalent antibody means that the antibody has two antigen-binding sites
  • trivalent antibody means that the antibody has three antigen-binding sites.
  • Native human immunoglobulin molecules usually have two antigen-binding sites, Fabs usually have a single antigen-binding site, and single variable domains, ScFvs usually have a single antigen-binding site.
  • antigenic determinant refers to a polypeptide molecule that specifically binds an antigenic determinant.
  • a particular antigen binding moiety may be a Fab, ScFv, single variable domain or other structure.
  • Antigenic determinant herein is synonymous with antigenic epitope.
  • first”, “second”, etc. used herein with respect to antigen-binding moieties, antigens, Fc polypeptides, peptide linkers, heavy chains, light chains, polypeptides, etc., are for the convenience of distinguishing when more than one moiety of each type is present. use. Unless expressly stated, the use of these terms is not intended to confer a particular order or orientation to the bispecific antibodies.
  • fused means that the components (eg, antigen binding portion, Fc polypeptide, etc.) are linked by peptide bonds, directly or via one or more peptide linkers.
  • some peptide linkers consist of 1 to 50 amino acids linked by peptide bonds, wherein the amino acids can be selected from the 20 naturally occurring amino acids; in a more preferred embodiment, 1 to 50 amino acids are selected from glycine, alanine, acid, proline, serine, asparagine, glutamine and lysine.
  • exemplary peptide linkers may be polyglycine (especially (Gly) 4 , (Gly) 5 ), poly(Gly-Ser), (Gly) 3 AsnGlySer(Gly) 2 , (Gly) 3 Cys(Gly) 4.
  • GlyProAsnGlyGly or those disclosed in Table 4 of patent application WO2019195535, etc.
  • Fc eg, an antigen-binding moiety to an Fc
  • it is typically fused via a hinge region (one form of a peptide linker).
  • the first antigen-binding portion is fused to one Fc polypeptide of the Fc domain via a first hinge
  • the second antigen-binding portion is fused to another Fc polypeptide of the Fc domain via a second hinge.
  • the first hinge and/or the second hinge may comprise amino acids of the hinge region of human IgG (such as IgG1, IgG4, etc.), and the hinge region of human IgG comprises a natural hinge region or a variant thereof.
  • variable domain refers to the domain of an antibody that is involved in the binding of the antibody to an antigen.
  • natural four-chain antibodies such as derived from humans, mice, etc.
  • VH heavy chain variable region
  • VL light chain variable region
  • only heavy chain antibodies derived from animals such as camelids or sharks have a single variable region. domain.
  • each variable domain of a native antibody consists essentially of four "framework regions” and three "complementarity determining regions.”
  • the four framework regions are respectively referred to as framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4); said framework regions are respectively referred to as complementarity-determining regions in the art and hereinafter (CDR) spaced apart.
  • CDR complementarity-determining regions
  • the general structure of a variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the general structure of the heavy chain variable region can be expressed as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
  • variable domains confer specificity to an antibody for an antigen by having an antigen-binding site.
  • CDR complementarity determining region
  • HVR hypervariable region
  • Natural four-chain antibodies usually contain six CDRs, three in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), and the other three in the light chain can be In the variable region, there are light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), respectively.
  • Heavy chain-only antibodies or single variable domains typically have three CDRs (CDR1, CDR2 and CDR3).
  • the basis for the "contact" definition of the CDRs is the analysis of available complex crystal structures.
  • the boundaries of the CDRs of the same antibody variable region obtained based on different methods may be different, that is, the CDR sequences of the same antibody variable region defined by different methods may be different. Therefore, when referring to an antibody defined by a specific CDR sequence defined by the division of the present invention, the scope of the antibody also covers an antibody defined by a CDR sequence converted to any other definition (such as Chothia, AbM definition, etc.).
  • FR framework region
  • Fab refers to a protein consisting of the VH and CH1 of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin.
  • Fab means a Fab in its native form or modified.
  • a Fab in a native form comprises a Fab heavy chain (VH-CH1, N to C-terminal) consisting of a heavy chain variable region and a constant region CH1. direction), and the Fab light chain (VL-CL, N to C-terminal direction) composed of the light chain variable region and the constant region CL.
  • the modified Fab can be, for example, a Fab that introduces amino acid substitutions in the CH1/CL domain or/and VH/VL domain.
  • the modified Fab can be a Fab that introduces amino acid substitutions in the CL domain.
  • the modified Fab may also be, for example, a Fab with a domain substitution.
  • the domain substitution is the substitution of VH/VL or/and CH1/CL in the Fab.
  • the Fab heavy chain in Fab refers to the polypeptide chain including VH
  • the Fab light chain refers to the polypeptide chain including VL.
  • the light chain constant region is a human kappa chain constant region.
  • the light chain constant region is a human lambda chain constant region.
  • the heavy chain constant region can be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, such as IgGl, IgG2, IgG3, and IgG4.
  • scFv includes the VH and VL domains of an immunoglobulin, wherein these domains are present in a single polypeptide chain.
  • the scFv further comprises a peptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.
  • Fc domain a region of an immunoglobulin heavy chain, which comprises at least part of the constant region.
  • the term includes native sequence Fc and variant Fc.
  • the C-terminal lysine (Lys447) of Fc may or may not be present.
  • the numbering of amino acid residues in the Fc or constant regions is according to the EU numbering system, also known as the EU index, described in Kabat, E.A et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
  • Fc polypeptides refers to one of the two polypeptides forming a dimeric Fc domain.
  • the Fc polypeptide of an IgG Fc domain comprises IgG CH2 and IgG CH3 constant regions.
  • effector functions refers to those biological activities attributable to the Fc domain of an antibody, which vary with the antibody isotype.
  • antibody effector functions are eg: Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and the like.
  • KD refers to the equilibrium dissociation constant expressed in molarity (M).
  • M molarity
  • the KD value of an antibody can be determined using methods known in the art. For example, one method of determining the KD of an antibody is using surface plasmon resonance, such as using a biosensor system, such as the Biacore system.
  • treatment refers to an attempt to alter the natural course of disease in an individual, and may be clinical intervention for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, amelioration or palliation of the disease state, and regression or improved prognosis.
  • subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, ovines, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.
  • the subject according to the invention is a human.
  • the terms "patient” or “subject” are used interchangeably.
  • isolated means that the compound of interest (eg, antibody or nucleic acid, etc.) has been separated from its natural environment.
  • Percent identity (%) of an amino acid sequence refers to after aligning the sequence to be compared with the specific amino acid sequence shown herein and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without Where any conservative substitutions are considered as part of the sequence identity, the percentage of amino acid residues in the aligned sequences that are identical to the amino acid residues of the particular amino acid sequence shown herein.
  • the identity alignment of amino acid sequences can be performed by various methods within the skill in the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • amino acid substitutions are expressed as: original amino acid-position-substituted amino acid, using three-letter codes or single-letter codes, including codes Xaa and X for amino acid residues.
  • H435R means substitution of amino acid H at position 435 with amino acid R; more than 1 may be included for substituted amino acids, eg T366Y/W means substitution of amino acid T at position 366 with amino acid Y or W .
  • Xn and Xaa are equivalent and refer to unspecified amino acids (Unspecified Amino Acid), and the scope covered by them is specified by the subsequent definitions in the relevant expressions.
  • the present invention provides bispecific antibodies with many desirable properties for targeted therapy. For example, it has one or more of the following characteristics:
  • the bispecific antibody can specifically bind to CD47 (such as human CD47) and PD-L1 (such as human PD-L1), and has good affinity; by binding to CD47, it can block the binding of CD47 and SIRP ⁇ , Promote macrophage-mediated phagocytosis of CD47-expressing tumor cells; by binding to PD-L1, it can inhibit the combination of PD-1 on T cells and PD-L1 on the surface of tumor cells, and induce T cell activation to exert anti-tumor effects ;
  • CD47 such as human CD47
  • PD-L1 such as human PD-L1
  • the bispecific antibody can specifically bind to CD47 (such as human CD47) and PD-L1 (such as human PD-L1), and has good affinity; by binding to CD47, it can block the binding of CD47 and SIRP ⁇ , Promote macrophage-mediated phagocytosis of CD47-expressing tumor cells; by binding to PD-L1, it can inhibit the combination of PD-1 on T cells and PD
  • the bispecific antibody does not cause substantial red blood cell reduction, anemia or red blood cell agglutination, and has good safety;
  • the bispecific antibody has good stability
  • the bispecific antibody has excellent anti-tumor effect. Some experiments have shown that the bispecific antibody has excellent anti-tumor effects on tumors such as human lymphoma, melanoma and the like.
  • the present invention provides a bispecific antibody, which comprises a first antigen-binding portion that binds to CD47 and a second antigen-binding portion that binds to PD-L1; wherein the first antigen-binding portion comprises:
  • a heavy chain variable region comprising:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4 or 5,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9 or 10, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, 12, 13, 14 or 15;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, 17, 18, 19 or 20,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25, and
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 26, 27, 28, 30 or 32.
  • the first antigen binding moiety comprises:
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26.
  • the first antigen binding moiety comprises:
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.
  • the first antigen binding moiety comprises:
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:13; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28.
  • the first antigen binding moiety comprises:
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:14; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32.
  • the first antigen binding moiety comprises:
  • a heavy chain variable region comprising: HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:10, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:15; and
  • a light chain variable region comprising: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30.
  • the first antigen binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising SEQ ID NO: 33, 35, 37, 39, 41, 83, HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in 85 or 87, the light chain variable region comprising SEQ ID NO: 34, 36, 38, 40, 42, 84, 86 or 88 of the variable region sequence LCDR1, LCDR2, and LCDR3.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 33, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:34.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 35, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:36.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 37, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:38.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 39, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:40.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 41, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:42.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 83, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:84.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 85, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:86.
  • the heavy chain variable region of the first antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 87, and the light chain of the first antigen-binding portion
  • the variable region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in SEQ ID NO:88.
  • the heavy chain variable region of the first antigen binding portion comprises at least 80%, 85% of the sequence shown in SEQ ID NO: 33, 35, 37, 39, 41, 83, 85 or 87 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids sequence.
  • the light chain variable region of the first antigen binding portion comprises at least 80%, 85% of the sequence shown in SEQ ID NO: 34, 36, 38, 40, 42, 84, 86 or 88 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids sequence.
  • the heavy chain variable region of the first antigen binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 33 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 34 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO: HCDR3 comprising the amino acid sequence shown in 11, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26.
  • the heavy chain variable region of the first antigen binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 35 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 36 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO HCDR3 comprising the amino acid sequence shown in 13, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the first antigen binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 37 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 38 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 2, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO HCDR3 comprising the amino acid sequence shown in 12, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.
  • the heavy chain variable region of the first antigen binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 39 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 40 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO: HCDR3 comprising the amino acid sequence shown in 14, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32.
  • the heavy chain variable region of the first antigen-binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 41 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable
  • the region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 42 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO: HCDR3 comprising the amino acid sequence shown in 15, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30.
  • the heavy chain variable region of the first antigen binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO:83 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 84 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO: HCDR3 comprising the amino acid sequence shown in 14, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 32.
  • the heavy chain variable region of the first antigen-binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO:85. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 86 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO HCDR3 comprising the amino acid sequence shown in 13, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the first antigen-binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 87 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the first antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 88 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the first antigen-binding portion further comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, comprising HCDR2 comprising the amino acid sequence shown in SEQ ID NO: HCDR3 comprising the amino acid sequence shown in 15, LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25 and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 33
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : Amino acid sequence shown in 34.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 35
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : Amino acid sequence shown in 36.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 37
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : Amino acid sequence shown in 38.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 39
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : Amino acid sequence shown in 40.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 41
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : The amino acid sequence shown in 42.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 83
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : The amino acid sequence shown in 84.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 85
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : The amino acid sequence shown in 86.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 87
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : The amino acid sequence shown in 88.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 81
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : The amino acid sequence shown in 82.
  • the heavy chain variable region of the first antigen-binding portion comprises the amino acid sequence shown in SEQ ID NO: 79
  • the light chain variable region of the first antigen-binding portion comprises SEQ ID NO : The amino acid sequence shown in 80.
  • the heavy chain variable region and the light chain variable region of the first antigen-binding portion comprise a sequence selected from the group consisting of SEQ ID NO: 43/44, 45/46, 47/48, 49/ 50, 51/52, 53/54, 55/56, 57/58, 59/60, 61/62, 63/64, 65/66, 67/68, 69/70, 71/72, 73/74, Amino acid sequences shown in 75/76, 77/78.
  • the first antigen binding moiety is murine or humanized. Humanization can reduce immunogenicity. In one example, the first antigen-binding portion is humanized.
  • Table S1 and Table S2 provide some exemplary CD47 binding monoclonal antibodies such as chimeric versions of 2B2, 2H8, 3F10, 16E5 and 14A9 antibodies (Xi2B2, Xi2H8, Xi3F10, Xi16E5 and Xi14A9) and human antibodies of 3F10, 16E5, 14A9
  • Table S3 provides the variable region sequences of these exemplary mAbs.
  • These exemplary antibodies can be used to construct the bispecific antibodies described herein.
  • the CDRs of the first antigen-binding portion of the bispecific antibody are selected from the six CDRs of any exemplary CD47-binding mAb.
  • the variable region of the first antigen-binding portion of the bispecific antibody is selected from the heavy chain variable region and light chain variable region of any exemplary CD47-binding mAb.
  • the second antigen-binding portion provides the ability to bind PD-L1, for example, it can bind primate such as human PD-L1.
  • the second antigen binding moiety is murine, humanized or fully human.
  • both the first antigen-binding portion and the second antigen-binding portion are humanized; in another example, the first antigen-binding portion is humanized, and the second antigen-binding portion is fully human of.
  • the second antigen binding portion comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, comprising SEQ ID NO: HCDR2 of the amino acid sequence shown in 200, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 201
  • the light chain variable region comprises: LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 202, comprising SEQ ID NO: 203 LCDR2 of the amino acid sequence shown, and LCDR3 comprising the amino acid sequence of SEQ ID NO:204.
  • the heavy chain variable region of the second antigen-binding portion comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region sequence shown in SEQ ID NO: 205;
  • the light chain variable region comprises LCDR1, HCDR2 and LCDR3 of the light chain variable region sequence shown in SEQ ID NO:206.
  • the heavy chain variable region of the second antigen binding portion comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90% of the sequence shown in SEQ ID NO: 205 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the light chain of the second antigen binding portion is variable The region comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 206 Amino acid sequences that are %, 97%, 98%, 99% or 100% identical.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 205
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 206. amino acid sequence.
  • the bispecific antibody herein may have no Fc domain, and the first antigen-binding portion and the second antigen-binding portion are fused through a suitable linker.
  • the bispecific antibodies herein may have an Fc domain.
  • the Fc domain can prolong the half-life and provide functions such as Fc domain-related effectors.
  • the first antigen-binding portion or/and the second antigen-binding portion may be fused to the N-terminal or C-terminal of the Fc domain.
  • the first antigen binding portion and the second antigen binding portion may take any suitable structural form.
  • the first antigen-binding moiety and the second antigen-binding moiety can each independently be a Fab, ScFv, ScFab or other structures.
  • the bispecific antibody is multivalent, such as bivalent, tetravalent, etc.
  • the bispecific antibody is bivalent, that is, the first antigen-binding portion and the second antigen-binding portion each provide monovalent binding to the corresponding antigen.
  • the first antigen binding moiety and the second antigen binding moiety are both Fabs.
  • one of the first and second antigen binding moieties has a domain substitution that is
  • the heavy chain variable region and the light chain variable region replace each other, or/and
  • the constant structure regions CH1 and CL replace each other.
  • Such domain substitutions prevent mismatching of heavy and light chains from different Fabs, thereby increasing the yield and purity of bispecific antibodies in recombinant production.
  • the problem of heavy and light chain mismatches in bispecific antibody production is overcome by exchanging the variable and/or constant regions within the first or second antigen-binding portion of the bispecific antibody such that Fabs of different specificities do not have the same arrangement of domains and thus do not "swap" the light chains.
  • the domain is replaced by a heavy chain variable region and a light chain variable region such that the antigen binding portion comprises a peptide chain consisting of a light chain variable region and a heavy chain constant region and a peptide chain consisting of a heavy chain variable region.
  • the domains are replaced by the constant domains CH1 and CL, such that the antigen binding portion comprises a peptide chain consisting of a heavy chain variable region and a light chain constant region and a peptide chain consisting of a light chain variable region and a light chain constant region.
  • Peptide chain composed of heavy chain constant region.
  • the domain substitutions are heavy chain variable regions and light chain variable regions replacing each other, and constant domains CH1 and CL replacing each other.
  • the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.
  • the first antigen-binding moiety is fused at its C-terminus to the N-terminus of one of the Fc polypeptides of the Fc domain
  • the second antigen-binding moiety is fused at its C-terminus to the N-terminus of the Fc domain. N-terminus of another Fc polypeptide.
  • the first antigen-binding moiety is Fab and the first antigen-binding moiety is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the Fc polypeptides of the Fc domain
  • the second The antigen binding portion is a Fab and the second antigen binding portion is fused at the C-terminus of its Fab heavy chain to the N-terminus of another Fc polypeptide of the Fc domain.
  • the first antigen-binding portion is Fab and the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc polypeptides of the Fc domain
  • the The second antigen-binding part is Fab and the second antigen-binding part is fused to the N-terminus of another Fc polypeptide of the Fc domain at the C-terminus of its Fab heavy chain, wherein the constant structural regions CH1 and CL replace each other.
  • FIG. 1 A specific configuration is shown in FIG. 1 .
  • the Fc domain of a bispecific antibody consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule.
  • the Fc domain of an immunoglobulin G (IgG) molecule is a dimer with each Fc polypeptide comprising CH2 and CH3 IgG heavy chain constant regions.
  • the two Fc polypeptides of the Fc domain are capable of stably associating with each other.
  • said bispecific antibody comprises an Fc domain.
  • the Fc domain of the multispecific antibody is an IgG Fc domain.
  • the Fc domain is an IgG1 Fc domain.
  • the Fc domain is an IgG4 Fc domain.
  • the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P, which amino acid substitution reduces the Fab arm exchange of IgG4 antibodies in vivo.
  • the Fc domain is a human Fc domain.
  • the Fc domain is a human IgG1 Fc domain.
  • the Fc domain comprises modifications, such as amino acid substitutions.
  • the modification can be, for example, a modification that promotes heterodimerization, a modification that changes effector function, or a modification that changes the ability to bind to protein A, etc.
  • the Fc comprises a modification that promotes heterodimerization.
  • the bispecific antibody comprises a different antigen-binding portion fused to one or the other of the two Fc polypeptides in the Fc domain, so that the two Fc polypeptides are usually contained in two different polypeptides in the chain. Recombinant co-expression of these polypeptides and subsequent dimerization yields several possible combinations of the two polypeptides.
  • the Fc domain comprises amino acid substitutions that facilitate association of the two Fc polypeptides of the Fc domain.
  • the site of the most extensive protein-protein interaction between two Fc polypeptides of the human IgG Fc domain is in the CH3 domain of the Fc domain.
  • the amino acid substitution is in the CH3 domain of the Fc domain.
  • the amino acid substitution is a so-called "knob-into-hole", comprising a “knob” amino acid substitution in one of the two Fc polypeptides of the Fc domain and two Fc A "hole” amino acid substitution in another of the polypeptides.
  • this method involves introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding depression ("hole”) at the interface of the other Fc polypeptide, so that the protrusion can be positioned in the depression to Promotes heterodimer formation and hinders homodimer formation.
  • Protrusions are constructed by replacing small amino acid side chains from the interface of an Fc polypeptide with larger side chains (eg, tyrosine or tryptophan, etc.). Complementary depressions of the same or similar size as the protrusions are created in the interface of another Fc polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine, etc.).
  • amino acid residues are substituted with amino acid residues having a larger side chain volume, whereby within the CH3 domain of the Fc polypeptide producing a bulge that can be positioned in a recess within the CH3 domain of another Fc polypeptide in which amino acid residues are replaced with amino acid residues having a smaller side chain volume, whereby the Fc A recess is created within the CH3 domain of the polypeptide.
  • one of the Fc polypeptides of the Fc domain comprises T366Y/W or/and S354C, and the other Fc polypeptide comprises Y407T/V, Y349C, T366S or/and L368A.
  • one of the Fc polypeptides of the Fc domain comprises amino acid substitutions T366Y/W and S354C, and the other Fc polypeptide comprises amino acid substitutions Y407T/V, Y349C, T366S and L368A.
  • the Fc may be the Fc of human IgG1.
  • the Fc domain comprises modifications that alter effector function.
  • the Fc domain of the bispecific antibody is modified to reduce the binding affinity and/or effector function of the Fc domain to Fc receptors compared to the Fc without the modification. Reducing the binding affinity of the Fc domain to Fc receptors and/or effector function is beneficial for improving cytokine release or/and side effects.
  • the modification that reduces the binding affinity of the Fc domain to an Fc receptor and/or reduces effector function is an amino acid substitution.
  • the Fc domain comprises an amino acid substitution at one or more positions selected from E233, L234, L235, N297, P331 and P329.
  • the Fc domain comprises an amino acid substitution at one or more positions selected from L234, L235, and P329.
  • the Fc domain comprises amino acid substitutions L234A and L235A.
  • the Fc domain is IgG1 Fc, especially human IgG1 Fc.
  • the Fc domain comprises an amino acid substitution at position P329.
  • the amino acid substitution is P329A, P329R or P329G.
  • the Fc domain comprises an amino acid substitution at position P329 and another amino acid substitution at a position selected from E233, L234, L235, N297, and P331.
  • the other amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S.
  • the Fc domain comprises amino acid substitutions at positions P329, L234, and L235.
  • the Fc domain comprises amino acid substitutions L234A, L235A and P329G ("P329G LALA").
  • the Fc domain comprises the amino acid substitutions L234A, L235A and P329A ("P329A LALA").
  • the Fc is IgG1 Fc, especially human IgG1 Fc.
  • the Fc domain is a human IgGl Fc domain comprising amino acid substitutions L234A, L235A, and P329A/G/R. In some specific embodiments, said Fc domain is a human IgGl Fc domain comprising amino acid substitutions L234A, L235A.
  • the Fc domain comprises a modification that reduces or eliminates binding of the CH3 region of an Fc polypeptide in the Fc domain to Protein A from Staphylococcus aureus.
  • the modification is an amino acid substitution.
  • the Fc domain comprises amino acid substitutions H435R and/or Y436F that occur only on one Fc polypeptide and not on the other Fc polypeptide.
  • said Fc domain comprises the amino acid substitution H435R or/and Y436F occurring in only one of the Fc polypeptides.
  • the Fc is IgG1 Fc, especially human IgG1 Fc.
  • the Fc domain in the bispecific antibody, may comprise a modification that promotes heterodimerization, a modification that changes effector function, or reduces or eliminates the CH3 region and A of one Fc polypeptide in the Fc.
  • the Fc domain comprises a modification that promotes association of two Fc polypeptides, a modification that reduces the binding affinity of the Fc domain for an Fc receptor and/or reduces effector function, and reduces or eliminates the Fc domain. Modification of the binding of the CH3 region of an Fc polypeptide to protein A. For specific schemes of the above-mentioned different types of modification, combinations can be made.
  • the bispecific antibody comprises:
  • a first heavy chain comprising the heavy chain variable region of said first antigen binding portion
  • a first light chain comprising the light chain variable region of said first antigen binding portion
  • a second light chain comprising the light chain variable region of said second antigen binding portion.
  • the first heavy chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the sequence shown in SEQ ID NO: 207 , 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence
  • the first light chain comprising the sequence shown in SEQ ID NO: 208 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences
  • said The double chain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the sequence shown in SEQ ID NO: 209 , 97%, 98%, 99% or 100% identical amino acid sequence
  • the second light chain comprising the sequence shown in SEQ ID NO: 210 has at least 85%, 86%, 87%, 88%, 89%, Amino
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 207
  • the first light chain comprises the amino acid sequence shown in SEQ ID NO: 208
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 208.
  • the amino acid sequence shown in ID NO: 209, the second light chain comprises the amino acid sequence shown in SEQ ID NO: 210.
  • the bispecific antibody binds to CD47 (eg, human CD47) with a KD value of 1E-08M or less.
  • the bispecific antibody has the following binding affinity (K D ) for human CD47: between about 1E-11M to about 1E-08M, about 1E-10M to about 1E-08M, about 5E-09M to the range of about 1E-08M.
  • the bispecific antibody has the following binding affinity (K D ) for human CD47: about 1E-08M or less, about 5E-09M or less, about 3E-09M or less, about 1E-09M or less, or about 1E-10M or less.
  • the binding affinity KD of the bispecific antibody herein to CD47 is measured by Biacore.
  • the bispecific antibody binds to PD-L1 (eg, human PD-L1) with a KD value of 1E-09M or less.
  • the bispecific antibody has the following binding affinity (K D ) for human PD-L1: between about 1E-11M to about 1E-08M, about 1E-11M to about 1E-09M, about 5E -10M to about 1E-10M range.
  • the bispecific antibody has the following binding affinity (K D ) for human PD-L1: about 1E-09M or less, about 1E-10M or less, about 5E-10M or less , or about 1E-11M or less.
  • the binding affinity K D of the bispecific antibody herein to PD-L1 is measured by Biacore.
  • variants of bispecific antibodies are provided.
  • Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the bispecific antibody or by peptide synthesis. Such modifications are, for example, deletion of amino acid residues from within the amino acid sequence of an antibody, insertion of amino acid residues into said amino acid sequence, or/and substitution of amino acid residues in said amino acid sequence. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct so long as the final variant possesses the desired characteristics, eg antigen binding, eg tumor killing potency and the like.
  • the present invention provides a pharmaceutical composition, which comprises a bispecific antibody and further comprises one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers or other agents.
  • the pharmaceutical composition is suitable for various administration modes, one example is intravenous administration.
  • the invention provides an isolated nucleic acid comprising an encoding bispecific antibody of the invention.
  • the nucleic acid sequences of some bispecific antibodies are exemplarily listed in the sequence listing.
  • the present invention provides a vector comprising said nucleic acid.
  • the vector is a cloning vector; in other embodiments, the vector is an expression vector, a specific example, the expression vector is pcDNA3.1(+).
  • the expression vector may optionally be any expression vector capable of expressing the bispecific antibody described herein.
  • the present invention provides a host cell comprising said vector, which is an appropriate host cell for cloning or expressing a bispecific antibody.
  • the host cell is a prokaryotic cell.
  • the host cell is a eukaryotic cell.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of bispecific antibodies.
  • Mammalian cells are, for example, Chinese hamster (CHO) ovary cells, CHO-S cells, and the like.
  • the present invention provides a method for preparing a bispecific antibody, the method comprising: cultivating the host cell to express the bispecific antibody, and isolating and purifying the bispecific antibody in the system specific antibody.
  • the nucleic acid encoding the bispecific antibody is isolated and inserted into one or more vectors for further cloning or/and expression in host cells.
  • the nucleic acid can be obtained by various methods well known in the art, such as gene splicing and chemical synthesis.
  • the prepared bispecific antibody can be purified by known techniques such as affinity chromatography, size exclusion chromatography and the like.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, etc., and these will be apparent to those skilled in the art.
  • bispecific antibodies can be determined by any of a number of well-known analytical methods, such as size exclusion chromatography and the like.
  • the bispecific antibodies herein can be identified, screened or characterized for their physicochemical properties or/and biological activities by a variety of assays known in the art.
  • the present invention provides a method for reducing or inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of the bispecific antibody.
  • the present invention provides the use of the bispecific antibody or the pharmaceutical composition in preparing a drug for treating cancer.
  • the present invention provides a method for treating cancer in a subject, which comprises administering a therapeutically effective amount of the bispecific antibody or the pharmaceutical composition to the subject.
  • said cancer is leukemia, lymphoma, myeloma, ovarian cancer, breast cancer, endometrial cancer, colorectal cancer, colon cancer, rectal cancer, bladder cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, Prostate cancer, pancreatic cancer, gastric cancer, liver cancer, gallbladder cancer, bile duct cancer, esophagus cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine gland cancer, adrenal gland cancer, pituitary gland cancer, skin cancer, soft tissue cancer, Vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, and uterine cancer, glioblastoma, medulloblastoma, astrocytoma, glia tumor, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome,
  • the present invention provides a method for increasing T cell activation, said method comprising contacting T cells with said bispecific antibody.
  • the present invention provides a method for promoting phagocytosis of macrophages in a subject, the method comprising administering a therapeutically effective amount of the bispecific antibody to the subject.
  • the present invention also provides the following specific embodiments, but protection scope of the present invention is not limited thereto:
  • Embodiment 1 A bispecific antibody comprising a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds PD-L1; wherein the first antigen-binding portion comprises:
  • a heavy chain variable region comprising:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5, 1, 2, 3 or 4,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, 6, 7, 8 or 9, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 15, 11, 12, 13 or 14;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20, 16, 17, 18 or 19,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, 21, 22, 23 or 24, and
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 30, 26, 27, 28 or 32.
  • Embodiment 2 The bispecific antibody of embodiment 1, wherein said first antigen binding moiety comprises:
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 5,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 15;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 20,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and
  • LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 30;
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 6, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 11;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 16,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 21, and
  • LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26;
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 2,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 12;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and
  • LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 27;
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 3,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 8, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 13;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 18,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 23
  • LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 28;
  • HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4,
  • HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 9, and
  • HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 14;
  • a light chain variable region comprising:
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 19,
  • LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 24, and
  • LCDR3 comprising the amino acid sequence shown in SEQ ID NO:32.
  • Embodiment 3 A bispecific antibody comprising a first antigen binding portion that binds CD47 and a second antigen binding portion that binds PD-L1; wherein the first antigen binding portion comprises a heavy chain variable region and a light chain Variable region, the variable region of the heavy chain comprising HCDR1, HCDR2 and HCDR3 of the variable region sequence shown in SEQ ID NO: 87, 33, 35, 37, 39, 41, 83 or 85, the variable region of the light chain
  • the region comprises LCDR1, LCDR2 and LCDR3 of the variable region sequence set forth in SEQ ID NO: 88, 34, 36, 38, 40, 42, 84 or 86.
  • Embodiment 4 The bispecific antibody according to any one of embodiments 1-3, wherein the heavy chain variable region of the first antigen binding portion comprises a sequence corresponding to SEQ ID NO: 33, 35, 37, 39, Amino acid sequences having at least 80% identity to the sequence set forth in 41, 83, 85 or 87.
  • Embodiment 5 The bispecific antibody according to any one of embodiments 1-4, wherein the light chain variable region of the first antigen-binding portion comprises a sequence corresponding to SEQ ID NO: 34, 36, 38, 40, Amino acid sequences having at least 80% identity to the sequence set forth in 42, 84, 86 or 88.
  • Embodiment 6 The bispecific antibody according to any one of embodiments 1-5, wherein the heavy chain variable region of the first antigen binding portion comprises SEQ ID NO: 33, 35, 37, 39, 41
  • the amino acid sequence shown in , 83, 85 or 87, the light chain variable region of the first antigen-binding part comprises the amino acid sequence shown in SEQ ID NO: 34, 36, 38, 40, 42, 84, 86 or 88 .
  • Embodiment 7 The bispecific antibody according to any one of embodiments 1-6, wherein the heavy chain variable region and the light chain variable region of the first antigen binding portion are selected from any of the following: comprising :
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 33, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 34 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 35, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 36 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 37, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 38 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO:39, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO:40 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 41, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 42 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 83, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 84 Amino acid sequences with at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 85, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 86 amino acid sequences of at least 80% identity;
  • the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the sequence shown in SEQ ID NO: 87, and the light chain variable region comprises an amino acid sequence with the sequence shown in SEQ ID NO: 88 Amino acid sequences of at least 80% identity.
  • Embodiment 8 The bispecific antibody of embodiment 7, wherein the heavy chain variable region and the light chain variable region of the first antigen binding portion are selected from any of the following:
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 41, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 84;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 87, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 88;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 81, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 82; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 79, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 80.
  • Embodiment 9 The bispecific antibody according to any one of embodiments 1-8, wherein said second antigen binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises : HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 200, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 201; the light chain variable region comprises: comprising SEQ The LCDR1 of the amino acid sequence shown in ID NO: 202, the LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 203, and the LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 204.
  • the heavy chain variable region comprises : HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 199, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 200, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 201;
  • Embodiment 10 The bispecific antibody according to any one of embodiments 1-8, wherein the heavy chain variable region of the second antigen-binding portion comprises the heavy chain variable region shown in SEQ ID NO: 205 HCDR1, HCDR2 and HCDR3 of the sequence; the light chain variable region of the second antigen-binding portion comprises LCDR1, HCDR2 and LCDR3 of the light chain variable region sequence shown in SEQ ID NO: 206.
  • Embodiment 11 The bispecific antibody of embodiment 9 or 10, wherein the heavy chain variable region of the second antigen binding portion comprises amino acids at least 85% identical to the sequence set forth in SEQ ID NO: 205 sequence, the light chain variable region of the second antigen binding portion comprises an amino acid sequence with at least 80% identity to the sequence shown in SEQ ID NO: 206.
  • Embodiment 12 The bispecific antibody according to embodiment 11, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 205, and the light chain variable region comprises SEQ ID NO: The amino acid sequence shown in 206.
  • Embodiment 13 The bispecific antibody according to any one of embodiments 1-12, wherein said first and second antigen binding moieties are each independently a Fab, ScFv or ScFab.
  • Embodiment 14 The bispecific antibody of embodiment 13, wherein the first and second antigen binding moieties are both Fabs.
  • Embodiment 15 The bispecific antibody of embodiment 14, wherein one of said first and second antigen binding moieties has a domain substitution, said domain substitution being
  • the heavy chain variable region and the light chain variable region replace each other, or/and
  • the constant structure regions CH1 and CL replace each other.
  • Embodiment 16 The bispecific antibody according to embodiment 15, wherein in the second antigen-binding portion, the constant domains CH1 and CL replace each other such that the second antigen-binding portion comprises a heavy chain variable region and a light
  • Embodiment 17 The bispecific antibody according to any one of embodiments 1-16, wherein the bispecific antibody further comprises an Fc domain consisting of two Fc polypeptides.
  • Embodiment 18 The bispecific antibody of embodiment 17, wherein said first antigen binding portion is fused at its C-terminus to the N-terminus of one of the Fc polypeptides of said Fc domain, said second antigen binding portion The N-terminus of another Fc polypeptide fused at its C-terminus to the Fc domain.
  • Embodiment 19 The bispecific antibody according to embodiment 18, wherein said first antigen binding portion is a Fab and said first antigen binding portion is fused at the C-terminus of its Fab heavy chain to one of said Fc domains
  • Embodiment 20 The bispecific antibody according to any one of embodiments 17-19, wherein said Fc domain is an IgG Fc domain.
  • Embodiment 21 The bispecific antibody according to embodiment 20, wherein said IgG Fc domain is a human IgG Fc domain, preferably an Fc domain of human IgGl or human IgG4.
  • Embodiment 22 The bispecific antibody according to any one of embodiments 17-21, wherein said Fc domain comprises amino acid substitutions that facilitate association of the two Fc polypeptides of said Fc domain.
  • Embodiment 23 The bispecific antibody according to any one of embodiments 17-22, wherein the Fc domain comprises amino acid substitutions that reduce the binding affinity and/or effector function of the Fc domain for Fc receptors.
  • Embodiment 24 The bispecific antibody according to embodiment 23, wherein amino acid substitutions that reduce the binding affinity of the Fc domain to Fc receptors and/or effector function are in one or more of the group selected from the group according to EU numbering Locations: L234, L235 and P329.
  • Embodiment 25 The bispecific antibody according to any one of embodiments 17-24, wherein the Fc domain comprises amino acid substitutions that reduce or eliminate binding of the CH3 region of one of the Fc polypeptides in the Fc domain to Protein A.
  • Embodiment 26 The bispecific antibody according to any one of embodiments 1-25, wherein said bispecific antibody comprises:
  • a first heavy chain comprising the heavy chain variable region of said first antigen binding portion
  • a first light chain comprising the light chain variable region of said first antigen binding portion
  • a second light chain comprising the light chain variable region of said second antigen binding portion.
  • Embodiment 27 The bispecific antibody of embodiment 26, wherein said first heavy chain comprises an amino acid sequence at least 85% identical to the sequence set forth in SEQ ID NO: 207, said first light chain comprises An amino acid sequence having at least 85% identity to the sequence shown in SEQ ID NO: 208, said second heavy chain comprising an amino acid sequence having at least 85% identity to the sequence shown in SEQ ID NO: 209, said second light The chain comprises an amino acid sequence at least 85% identical to the sequence shown in SEQ ID NO:210.
  • Embodiment 28 The bispecific antibody according to any one of embodiments 1-27, which is bivalent.
  • Embodiment 29 The bispecific antibody according to any one of embodiments 1-28, wherein said bispecific antibody is chimeric or humanized.
  • Embodiment 30 The bispecific antibody of any one of embodiments 1-29, wherein said bispecific antibody binds human CD47 with a KD ⁇ 1E-08M.
  • Embodiment 31 The bispecific antibody according to any one of embodiments 1-30, wherein said bispecific antibody binds human PD-L1 with a KD ⁇ 1E-09M.
  • Embodiment 32 An isolated nucleic acid encoding the bispecific antibody according to any one of embodiments 1-31.
  • Embodiment 33 A vector comprising the nucleic acid according to embodiment 32.
  • Embodiment 34 A host cell comprising the nucleic acid of embodiment 32, or the vector of embodiment 33.
  • Embodiment 35 A method of producing the bispecific antibody of any one of embodiments 1-31, comprising culturing the host cell of embodiment 34 to express the bispecific antibody, and The bispecific antibody is separated and purified in the system.
  • Embodiment 36 A pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1-31 and a pharmaceutically acceptable carrier.
  • Embodiment 37 A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the bispecific antibody of any one of embodiments 1-31 or the bispecific antibody of embodiment 36. said pharmaceutical composition.
  • Embodiment 38 The method of treating cancer in a subject according to embodiment 37, wherein the cancer is leukemia, lymphoma, myeloma, ovarian cancer, breast cancer, endometrial cancer, colorectal cancer, bladder cancer Cancer, urothelial cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, esophagus cancer, renal cell carcinoma, thyroid cancer, head and neck cancer, testicular cancer, endocrine adenocarcinoma , adrenal gland cancer, pituitary gland cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, and uterine cancer, glioblast medulloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma
  • hCD47-His Tag protein (ACRObiosystems, catalog number: CD7-H5227 ) (50 ⁇ g/mouse Balb/c) or hCD47-mFc (ACRObiosystems, catalog number: CD7-H52A5) to subcutaneously immunize Balb/c mice every 2 weeks, three times for 6 weeks.
  • hCD47-mFc (ACRObiosystems, catalog number: CD7-H52A5) protein was used, 20 ⁇ g/mouse, intramuscularly injected (aqueous phase adjuvant). Three days before cell fusion, mice were boosted by intravenous injection of antigen without adjuvant.
  • Splenocytes (1 ⁇ 10 8 ) from immunized mice were fused with SP2/0 myeloma cells (1.5 ⁇ 10 7 ) using polyethylene glycol 1500 (Polyethylene Glycol 1500; Roche, catalog number: 10783641001). After confluency, cells were dispensed into 96-well plates at 0.1 mL/well, and incubated in an incubator at 37°C, 5% CO 2 . On day 1, cells were fed by adding an additional 0.1 mL per well of HAT medium containing serum and supplemented with 2 ⁇ methotrexate. On days 3 and 7, replace 0.1 mL of medium from each well with 0.1 mL of fresh HAT medium. Screening was typically performed between days 9-14 and positive wells were screened by testing culture supernatants by ELISA for antibodies reactive with hCD47-His Tag (ACRObiosystems, Cat. No.: CD7-H5227).
  • ACRObiosystems Cat. No.: CD7-H5227
  • Hybridoma cells were cloned by limiting dilution method.
  • the selected positive mixed clones were limitedly diluted, and one positive mixed clone was spread in two 96-well plates (100cells/plate, 200cells/plate), and the hybridoma cells were cultured in RPMI 1640 (Hyclone company, catalog number: SH30809.01). After 10 days, the subclones after limited dilution were also tested by ELISA to detect the culture supernatant, and positive subclones were picked. The cloned hybridoma cells were kept and used for cDNA extraction.
  • RNA was isolated from the screened hCD47 antibody-producing hybridoma cell lines using an RNA extraction kit ((Takara, catalog number: 9767), using total RNA as a template, and using a reverse transcription kit (Thermo Fisher, catalog #: K1652) synthesized first-strand cDNA. The synthesized cDNA was then used for in vitro amplification of antibody-related sequences by PCR reactions using degenerate mouse IgG primers.
  • PCR amplification products were separated by electrophoresis in 1% agarose/Tris-acetate gel.
  • a DNA fragment of the expected size (approximately 450 bp for heavy and light chains) was excised from the gel and purified.
  • 3 ⁇ L of the purified PCR product was cloned into pMD-19T vector (Takara, catalog number: 6013), and transformed into DH5 ⁇ chemically competent E. coli (Takara, catalog number: 9057). In each ligation reaction, 10 positive clones were randomly selected for DNA sequencing using the M13 forward primer.
  • VH heavy chain variable regions
  • 16E5 16E5
  • VL light chain variable region
  • the VL region of each mouse antibody is connected to the human ⁇ chain constant region to construct a chimeric light chain
  • the mouse VH region is connected to human IgG4 (EU number S228P ) constant region to construct a chimeric heavy chain.
  • the expression plasmids of the chimeric light chain and the expression plasmid of the chimeric heavy chain were respectively constructed by conventional genetic engineering methods, and CHO cells (200mL system, at 10 6 cells/mL) were used with 100 ⁇ g of each chimeric heavy chain expression plasmid and chimeric light chain expression plasmids were transfected and cultured for 6 days. The chimeric antibody in the supernatant was then purified using a protein A column.
  • Xi2H8 chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 89-90; chimeric heavy chain and its encoding nucleic acid: Chimeric light chain and its encoding nucleic acid: SEQ ID NOs:91-92
  • Xi2B2 chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs:97-98; chimeric light chain and its encoding nucleic acid:99 -100
  • Xi3F10 chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 101-102; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 103-104
  • Xi16E5 chimeric heavy chain and its encoding Nucleic acid: SEQ ID NOs: SEQ ID NOs: SEQ ID NOs: 103-104
  • Xi16E5 chimeric heavy chain and its encoding Nucleic acid: SEQ ID NOs:
  • Antibody humanization design :
  • Human-mouse chimeric antibodies to 3F10, 14A9, and 16E5 were humanized using the CDR grafting method (see, eg, US Patent No. 5,225,539).
  • CHO cells (200 mL system at 10 cells/mL) were transfected with 100 ⁇ g of each humanized antibody heavy chain expression plasmid and light chain expression plasmid and incubated with ExpiCHO medium (Gibco; Cat. No.: A29100 -01) Culture in an incubator at 37°C and 5% CO 2 for 6 days. After the supernatant was obtained by centrifugation, the humanized antibody in the supernatant was purified by protein A column.
  • ExpiCHO medium Gibco; Cat. No.: A29100 -01
  • Human CD47 protein Human CD47 Protein, His Tag; ACRObiosystems, catalog number: CD7-H5227
  • cynomolgus monkey CD47 protein Cynomolgus/Rhesus macaque CD47 Protein, His Tag; ACRObiosystems, catalog number: CD7-C52H1
  • Biacore analysis was performed at 25°C and at a data collection rate of 1 Hz Make a note.
  • Polyclonal rabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10 mM sodium acetate pH 5.0, and immobilized to the reference flow cell and About 15000RM was reached on the experimental flow cell. At the beginning of each cycle, diluted test antibody (1.5 ⁇ g/mL) was injected over the experimental flow cell for 1 min to be captured.
  • anti-CD47 antibody to promote phagocytosis of tumor cells by macrophages containing SIRP ⁇ on the cell surface was determined based on flow cytometry.
  • Mononuclear cells were isolated from human peripheral blood mononuclear cells (PBMC, Saili Biology, catalog number: 190056) using a mononuclear cell isolation kit (STEMCELL, catalog number: 19058), and spread into 24-well plates (Greiner bio -one, catalog number: 662-160), 5 ⁇ 10 5 cells per well, and induced in the environment of 100ng/mL stimulating factor M-CSF (R&D Systems company, catalog number: 216-MC-010) for 7 days , thereby inducing macrophages.
  • PBMC peripheral blood mononuclear cells
  • STEM mononuclear cell isolation kit
  • the HL60 cells were washed three times with PBS buffer, added to the macrophages, and reacted in a 37°C, 5% CO 2 incubator for 2-4 hours. After washing three times with PBS buffer, 260 ⁇ L PBS and 20 ⁇ L anti-APC- CD14 (APC Mouse Anti-Human CD14, BD Company, catalog number: 555399) and 20 ⁇ L PE-CD11b (PE Mouse Anti-Human CD11b/Mac-1, BD Company, catalog number: 555388) antibody mixture, 4 ° C Incubate with light for 30 min, wash with PBS buffer three times, digest with trypsin (Gibco, catalog number: 12604-013) and detect the phagocytosis rate (%) of macrophages with a flow cytometer (BD Company, model specification: C6).
  • PBS buffer 20 ⁇ L anti-APC- CD14
  • PE-CD11b PE Mouse Anti-Human CD11b/Mac-1, BD Company, catalog number: 555
  • anti-CD47 chimeric antibodies and anti-CD47 humanized antibodies on macrophage phagocytosis are shown in Table 3 and Table 4, among which chimeric antibodies Xi3F10, Xi16E5, Xi14A9 and anti-CD47 humanized antibodies have good phagocytosis-promoting effects, Humanized antibodies hz14A9-2.3 and hz14A9-2.4 performed better.
  • Table 3 Analysis of anti-CD47 chimeric antibody based on promoting macrophage phagocytosis (phagocytosis rate, %)
  • Table 4 Analysis of anti-CD47 humanized antibody based on promoting macrophage phagocytosis (phagocytosis rate, %)
  • the effect of anti-CD47 antibody on CD4+ T cells was determined by flow cytometry-based assay method.
  • T cell isolation kit (Miltenyi Biotech, catalog number: 130-094-131) to isolate CD4+ T cells from human peripheral blood mononuclear cells (PBMC, Saili Biotech, catalog number: 190056), and spread into 24 Orifice plate (Greiner bio-one, catalog number: 662106), 5 ⁇ 10 5 cells per well, and anti-CD47 antibodies were added at the same time (see Table 5 for chimeric antibodies, and Table 6 for humanized antibodies) to make the final concentration 10 ⁇ g/mL and 1 ⁇ g/mL, respectively, added 1640 complete medium, cultured in 37 ° C, 5% CO 2 incubator for 20 h, used apoptosis kit (STEMCELL, catalog number: 556547) to stain at room temperature for 15 min, flow cytometer ( BD, model specification: C6) Detect the apoptosis rate (%), and calculate the apoptosis rate by the sum of early apoptosis and late apoptosis. The results showed that anti
  • CCRF-CEM-PDL1-20 CCRF-CEM cells expressing PDL1
  • Raji-PDL1 Raji cells expressing PDL1
  • flow cytometry Thermo, Thermo Attune NxT
  • PE Phycoerythrin Fluorescence Quantitation Kit BD biosciences, catalog number 340495
  • R-PE phycoerythrin fluorescently labeled PE anti-human PD-L1
  • PE anti-human CD47 Biolegend, catalog number 323108
  • Table 7 The number of PD-L1 and CD47 antigens on the cell surface
  • the constructed anti-PDL1*CD47 bispecific antibody CRS147-3355b consists of four polypeptide chains, and the amino acid and nucleotide sequences of each polypeptide chain are shown in Table 8.
  • the control antibody BM is composed of three polypeptide chains, and its sequence is derived from SEQ ID NO: 1, 8, and 14 in the patent application WO2020259605A1.
  • the transfected expression volume is 0.1-1L, and the cell density is 6E+06cells/ mL.
  • the cells were placed in ExpiCHO TM medium (manufacturer: Thermo, product number: A2910001), and cultured at 32°C, 5% CO 2 , and 130rpm for expression. On the 10th day, the cell supernatant was collected and used The AKTA Pure 25L system and MabSelect SuRe LX medium were used for protein A affinity chromatography of the target protein. After elution, NanoDrop Lite (Thermo Fisher Scientific) was used to measure the protein concentration of the eluted harvest, and SEC-HPLC was used to detect the purity of the eluted protein.
  • the antibody molecule was analyzed by SDA-PAGE, molecular weight detection, mass spectrometry, and the like.
  • HZ5G11 monoclonal antibody (the amino acid sequence of the heavy chain and the light chain is SEQ ID NO: 215/216)
  • HZ14A9 monoclonal antibody (the amino acid sequence of the heavy chain and the light chain is SEQ ID NO: 216) was detected by Biacore 8K system. ID NO: 217/218), CRS147-3355b and affinity properties of BM bispecific antibodies.
  • HZ5G11 monoclonal antibody, HZ14A9 monoclonal antibody, CRS147-3355b and BM bispecific antibody were respectively immobilized on the sensor chip by capture method, and human PD-L1 with a certain concentration gradient was injected (manufacturer: Biopsies, product number: PD1 -H52H3) or human CD47 (manufacturer: Baipsies, item number: CD7-H5227) protein antigen solution, Biacore 8K Control Software 3.0 real-time collection of binding and dissociation curves, and data analysis by Biacore Insight Evaluation Software 3.0, the binding Rate constant K a , dissociation rate constant K d and equilibrium dissociation constant K D .
  • the thermal stability properties of HZ5G11 mAb, HZ14A9 mAb, CRS147-3355b and BM bispecific antibody were characterized by Nano DSF (Nano Temper). Adjust the concentration of the antibody sample to be tested to about 1.0mg/mL, then use a quartz capillary to absorb a small amount of sample, place it in the Nano DSF sample suspension, and program the temperature at a constant rate of 1.0°C/min in the range of 20.0°C-95.0°C , collect optical signal changes of protein samples, and analyze protein thermal stability.
  • the suspension cells Raji-PDL1 and CCRF-CEM-PDL1-20 were taken respectively, and the cells were collected by centrifugation at 1500rpm for 3min. Add PBS to resuspend the cells, count the cells, add 1.5E+05 cells/well to a 96-well plate, and centrifuge at 1500 rpm for 3 min.
  • CRS147-3355b and BM exhibited almost identical cell-binding behaviors.
  • CRS147-3355b and BM showed completely different cell binding behavior; compared with BM, CRS147-3355b had a better affinity for tumor cells.
  • MLR Mixed lymphocyte reaction
  • the control antibody was HZ5G11 (anti-PDL1 antibody), which had obvious effects on stimulating T cells to secrete IL-2 ( Figure 6A) and IFN- ⁇ ( Figure 6B).
  • Embodiment 11 Erythrocyte agglutination experiment
  • CRS147-3355b and BM were characterized by human erythrocyte agglutination assay.
  • the red blood cells are washed thoroughly to remove the plasma attached to the surface of the red blood cell membrane. Wash the red blood cells 3 times with isotonic diluent, centrifuge at 2000rpm/min for 5min for the first 2 times; centrifuge at 2000rpm/min for 10min for the last time, and discard the supernatant.
  • Red blood cells were made into 2% cell suspension using PBS, 100 ⁇ L was pipetted into a 96-well U-shaped plate, centrifuged at 1500 rpm/min for 5 min, and the supernatant was discarded.

Abstract

提供了一种针对免疫检查点的双特异性抗体。具体地提供了双特异性抗体、编码它们的核酸、包含该核酸的载体、包含该载体的细胞、包含它们的药物组合物，还提供了其治疗相关疾病的受试者的用途。

Description

针对免疫检查点的双特异性抗体 技术领域
本发明涉及双特异性抗体，尤其涉及靶向CD47和PD-L1的双特异性抗体。
背景技术
CD47是一种广泛表达于多个物种和各个组织之间的跨膜糖蛋白，也被称为整合素相关蛋白，是免疫球蛋白超家族中的成员。抑制性受体信号调节蛋白α(SIRPα)是CD47的配体之一，CD47结合在SIRPα的NH 2末端IgV样结构域。SIRPα主要表达于髓系来源的细胞，包括巨噬细胞、粒细胞、DC细胞、肥大细胞以及它们的前体，包括造血干细胞。CD47表达和/或活化已经牵涉许多疾病和病症。不同的研究表明，几乎所有的肿瘤细胞和肿瘤组织都高表达CD47。肿瘤细胞表面高表达的CD47通过与巨噬细胞表面SIRPα结合，释放“别吃我”(don’t eat me)的信号，从而导致肿瘤组织浸润区的巨噬细胞不但同肿瘤细胞和谐相处，而且还会通过促进肿瘤内血管增殖，抑制效应T细胞发挥作用，促进肿瘤细胞扩增和生长。
基于CD47靶点本身的特殊性，针对其的药物研发往往围绕着如何在起到恢复巨噬细胞对肿瘤细胞的吞噬作用的同时，也避免其对正常细胞的损伤的问题。目前由CD47引起的严重贫血反应仍然是针对该靶点药物应用的一大挑战。
程序性死亡分子1及其配体是一对负性免疫共刺激分子。PD-L1(Programmed death-ligand l，PD-L1或PDL1)又称为CD274和B7-H1，是程序性死亡分子l(Programmed death，PD-1)的一个配体。正常情况下，组织细胞表面的PD-L1与淋巴细胞表面的PD-1结合后，可抑制淋巴细胞功能，诱导活化的淋巴细胞凋亡，从而在自身免疫耐受及防止自身免疫性疾病中发挥重要作用。多种肿瘤细胞表面也表达PD-L1，肿瘤细胞表达的PD-L1，可与肿瘤浸润淋巴细胞表面的PD-1分子结合，抑制淋巴细胞的功能及细胞因子的释放，并诱导淋巴细胞凋亡，从而抵抗淋巴细胞的杀伤作用，最终导致肿瘤发生免疫逃逸。靶向程序性死亡配体1(PD-L1)的单抗，阻止PD-L1与T细胞表面的PD-1和B7-1受体结合，使T细胞恢复活性，从而增强免疫应答，具有治疗多种类型肿瘤的潜力。
已有一些靶向CD47和PD-L1的抗体被开发，但仍然是有限的，需要更多可用的选择。
发明内容
本发明提供了结合CD47和PD-L1的双特异性抗体。
一方面，本发明提供了双特异性抗体，其包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含：
重链可变区，其包含：
HCDR1，其包含SEQ ID NO：1、2、3、4或5所示的氨基酸序列，
HCDR2，其包含SEQ ID NO：6、7、8、9或10所示的氨基酸序列，和
HCDR3，其包含SEQ ID NO：11、12、13、14或15所示的氨基酸序列；和
轻链可变区，其包含：
LCDR1，其包含SEQ ID NO：16、17、18、19或20所示的氨基酸序列，
LCDR2，其包含SEQ ID NO：21、22、23、24或25所示的氨基酸序列，和
LCDR3，其包含SEQ ID NO：26、27、28、30或32所示的氨基酸序列。
在一些实施方案中，所述第一抗原结合部分包含：
(i)重链可变区，其包含：包含SEQ ID NO：1所示氨基酸序列的HCDR1，包含SEQ ID NO：6所示氨基酸序列的HCDR2，和包含SEQ ID NO：11所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：16所示氨基酸序列的LCDR1，包含SEQ ID NO：21所示氨基酸序列的LCDR2，和包含SEQ ID NO：26所示氨基酸序列的LCDR3；
(ii)重链可变区，其包含：包含SEQ ID NO：2所示氨基酸序列的HCDR1，包含SEQ ID NO：7所示氨基酸序列的HCDR2，和包含SEQ ID NO：12所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：17所示氨基酸序列的LCDR1，包含SEQ ID NO：22所示氨基酸序列的LCDR2，和包含SEQ ID NO：27所示氨基酸序列的LCDR3；
(iii)重链可变区，其包含：包含SEQ ID NO：3所示氨基酸序列的HCDR1，包含SEQ ID NO：8所示氨基酸序列的HCDR2，和包含SEQ ID NO：13所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：18所示氨基酸序列的LCDR1，包含SEQ ID NO：23所示氨基酸序列的LCDR2，和包含SEQ ID NO：28所示氨基酸序列的LCDR3；
(iv)重链可变区，其包含：包含SEQ ID NO：4所示氨基酸序列的HCDR1，包含SEQ ID NO：9所示氨基酸序列的HCDR2，和包含SEQ ID NO：14所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：19所示氨基酸序列的LCDR1，包含SEQ ID NO：24所示氨基酸序列的LCDR2，和包含SEQ ID NO：32所示氨基酸序列的LCDR3；或者
(v)重链可变区，其包含：包含SEQ ID NO：5所示氨基酸序列的HCDR1，包含SEQ ID NO：10所示氨基酸序列的HCDR2，和包含SEQ ID NO：15所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：20所示氨基酸序列的LCDR1，包含SEQ ID NO：25所示氨基酸序列的LCDR2，和包含SEQ ID NO：30所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区和轻链可变区选自以下任一项：包含：
(a)所述的重链可变区包含与SEQ ID NO：33所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：34所示序列具有至少80％同一性的氨基酸序列；
(b)所述的重链可变区包含与SEQ ID NO：35所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：36所示序列具有至少80％同一性的氨基酸序列；
(c)所述的重链可变区包含与SEQ ID NO：37所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：38所示序列具有至少80％同一性的氨基酸序列；
(d)所述的重链可变区包含与SEQ ID NO：39所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：40所示序列具有至少80％同一性的氨基酸序列；
(e)所述的重链可变区包含与SEQ ID NO：41所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：42所示序列具有至少80％同一性的氨基酸序列；
(f)所述的重链可变区包含与SEQ ID NO：83所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：84所示序列具有至少80％同一性的氨基酸序列；
(g)所述的重链可变区包含与SEQ ID NO：85所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：86所示序列具有至少80％同一性的氨基酸序列；以及
(h)所述的重链可变区包含与SEQ ID NO：87所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：88所示序列具有至少80％同一性的氨基酸序列。
在一些实施方案中，所述第二抗原结合部分包含重链可变区和轻链可变区，其中重链可变区包含：包含SEQ ID NO：199所示氨基酸序列的HCDR1、包含SEQ ID NO：200所示氨基酸序列的HCDR2、和包含SEQ ID NO：201所示氨基酸序列的HCDR3，轻链可变区包含：包含SEQ ID NO：202所示氨基酸序列的LCDR1、包含SEQ ID NO：203所示氨基酸序列的LCDR2、和包含SEQ ID NO：204所示氨基酸序列的LCDR3。
在一些实施方案中，所述第二抗原结合部分的重链可变区包含与SEQ ID NO：205所示序列具有至少85％同一性的氨基酸序列，所述第二抗原结合部分的轻链可变区包含与SEQ ID NO：206所示序列具有至少80％同一性的氨基酸序列。在一些具体的实施方案中，所述的重链可变区包含SEQ ID NO：205所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：206所示的氨基酸序列。
在一些实施方案中，所述双特异性抗体包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含：重链可变区，其包含：包含SEQ ID NO：5所示氨基酸序列的HCDR1，包含SEQ ID NO：10所示氨基酸序列的HCDR2，和包含SEQ ID NO：15所示氨基酸序列的HCDR3；和轻链可变区，其包含：包含SEQ ID NO：20所示氨基酸序列的LCDR1，包含SEQ ID NO：25所示氨基酸序列的LCDR2，和包含SEQ ID NO：30所示氨基酸序列的LCDR3；所述第二抗原结合部分包含重链可变区和轻链可变区，其中重链可变区包含：包含SEQ ID NO：199所示氨基酸序列的HCDR1，包含SEQ ID NO：200所示氨基酸序列的HCDR2，和包含SEQ ID NO：201所示氨基酸序列的HCDR3；轻 链可变区包含：包含SEQ ID NO：202所示氨基酸序列的LCDR1，包含SEQ ID NO：203所示氨基酸序列的LCDR2，和包含SEQ ID NO：204所示氨基酸序列的LCDR3。
在一些实施方案中，所述双特异性抗体包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含：重链可变区，其包含SEQ ID NO：79所示氨基酸序列；和轻链可变区，其包含SEQ ID NO：80所示氨基酸序列；所述第二抗原结合部分包含：重链可变区，其包含SEQ ID NO：205所示氨基酸序列；和轻链可变区，其包含SEQ ID NO：206所示氨基酸序列。
在一些实施方案中，双特异性抗体还包含由两个Fc多肽构成的Fc域。
在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分各自独立的为Fab、ScFv、ScFab(单链Fab)或其他结构。
在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分均为Fab。在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分的其中之一具有结构域替换，所述结构域替换为重链可变区和轻链可变区彼此替换，或/和恒定结构区CH1和CL彼此替换。在一些实施方案中，所述第一抗原结合部分为Fab且所述第一抗原结合部分在其Fab重链的C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分为Fab且所述第二抗原结合部分在其Fab重链的C端融合至所述Fc域的另一个Fc多肽的N端。
在一些具体的实施方案中，所述双特异性抗体包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分为Fab并包含：重链可变区，其包含：包含SEQ ID NO：5所示氨基酸序列的HCDR1，包含SEQ ID NO：10所示氨基酸序列的HCDR2，和包含SEQ ID NO：15所示氨基酸序列的HCDR3；和轻链可变区，其包含：包含SEQ ID NO：20所示氨基酸序列的LCDR1，包含SEQ ID NO：25所示氨基酸序列的LCDR2，和包含SEQ ID NO：30所示氨基酸序列的LCDR3；所述第二抗原结合部分为Fab并包含重链可变区和轻链可变区，其中重链可变区包含：包含SEQ ID NO：199所示氨基酸序列的HCDR1，包含SEQ ID NO：200所示氨基酸序列的HCDR2，和包含SEQ ID NO：201所示氨基酸序列的HCDR3；轻链可变区包含：包含SEQ ID NO：202所示氨基酸序列的LCDR1，包含SEQ ID NO：203所示氨基酸序列的LCDR2，和包含SEQ ID NO：204所示氨基酸序列的LCDR3。在一些这些实施方案中，所述第一抗原结合部分和第二抗原结合部分的其中之一具有结构域替换，在所述第二抗原结合部分中，恒定结构区CH1和CL彼此替换，使得第二抗原结合部分包含由重链可变区和轻链恒定区构成的肽链和由轻链可变区和重链恒定区构成的肽链。在一些上述任一实施方案中，所述双特异性抗体还包含由两个Fc多肽构成的Fc域。在一些上述任一实施方案中，所述所述Fc域包含促进所述Fc域的两个Fc多肽相缔合的氨基酸取代，或/和所述Fc域包含降低Fc域对Fc受体的结合亲和力和/或效应子功能的氨基酸取代，或/和所述Fc域包含减少或消除Fc域中的一个Fc多肽的CH3区与A蛋白的结合的氨基酸取代。
在一些实施方案中，所述双特异性抗体包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分为Fab并包含：重链可变区，其包含SEQ ID NO：79所示氨基酸序列；和轻链可变区，其包含SEQ ID NO：80所示氨基酸序列；所述第二抗原结合部分为Fab并包含：重链可变区，其包含SEQ ID NO：205所示氨基酸序列；和轻链可变区，其包含SEQ ID NO：206所示氨基酸序列。在一些这些实施方案中，所述第一抗原结合部分和第二抗原结合部分的其中之一具有结构域替换，在所述第二抗原结合部分中，恒定结构区CH1和CL彼此替换，使得第二抗原结合部分包含由重链可变区和轻链恒定区构成的肽链和由轻链可变区和重链恒定区构成的肽链。在一些上述任一实施方案中，所述双特异性抗体还包含由两个Fc多肽构成的Fc域。在一些上述任一实施方案中，所述所述Fc域包含促进所述Fc域的两个Fc多肽相缔合的氨基酸取代，或/和所述Fc域包含降低Fc域对Fc受体的结合亲和力和/或效应子功能的氨基酸取代，或/和所述Fc域包含减少或消除Fc域中的一个Fc多肽的CH3区与A蛋白的结合的氨基酸取代。
在一些实施方案中，所述双特异性抗体由以下四条多肽链组成：
第一重链，其包含所述第一抗原结合部分的重链可变区；
第一轻链，其包含所述第一抗原结合部分的轻链可变区；
第二重链，其包含所述第二抗原结合部分的重链可变区；和
第二轻链，其包含所述第二抗原结合部分的轻链可变区。
在一些实施方案中，所述的双特异性抗体为二价的。
一方面，本发明提供了一种分离的核酸，其编码本发明所述的双特异性抗体。
一方面，本发明提供了一种载体，其包含本发明所述的核酸。
一方面，本发明提供了一种宿主细胞，其包含本发明所述的核酸，或本发明所述的载体。
一方面，本发明提供了一种制备所述的双特异性抗体的方法，其包括所述的宿主细胞，使其表达所述的双特异性抗体，并且在体系中分离纯化所述双特异性抗体。
一方面，本发明提供了一种药物组合物，其包含本发明所述的双特异性抗体和药学可接受载体。
一方面，本发明提供了一种在受试者中治疗癌症的方法，其包括对所述受试者施用治疗有效量的所述的双特异性抗体或所述的药物组合物。
附图说明
图1为anti-PDL1*CD47双特异性抗体CRS147-3355b的结构示意图。
图2为对照双特异性抗体BM的结构示意图。
图3为利用分子排阻色谱(SEC)检测的双特异性抗体CRS147-3355b的纯度。
图4为anti-PDL1*CD47双特异性抗体CRS147-3355b与CCRF-CEM-PDL1-20细胞的结合。
图5为anti-PDL1*CD47双特异性抗体CRS147-3355b与Raji-PDL1细胞的结合。
图6A为MLR中不同浓度抗体下的IL-2的产生量；图6B为MLR中不同浓度抗体下的IFNγ的产生量。
图7为anti-PDL1*CD47双特异性抗体CRS147-3355b对红细胞的凝集效果。
发明详述
本文对本申请进行了示例性的实施方式的描述，但是本领域技术人员将理解的是本申请的保护范围并不限于此，而是可基于本申请的精神和构思进行各种修饰、修改或改变，这些修饰、修改或改变后的内容仍然落在本申请的范围内。
术语
术语“抗体”指的是包含抗原结合位点的蛋白，涵盖各种结构的天然抗体和人工抗体，包括但不限于单克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体、三特异性抗体等)、单链抗体等。
术语“多特异性”意指抗体能够特异性结合多种不同的抗原决定簇，例如能够特异性结合两种或两种以上不同的抗原决定簇。通常，双特异性抗体包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。不同的抗原决定簇可以在相同或不同的细胞上表达。不同的抗原决定簇可以因抗原种类不同而不同(例如结合抗原CD47和PDL1)，也可以存在同一种抗原上。抗原决定簇是抗原物质分子表面或其他部位，具有一定组成和结构的特殊化学基团，能与其相应抗体或致敏淋巴细胞发生特异性结合的结构。抗原决定簇一个例子是，CD47作为一种抗原物质，其具有多种结构已确定或结构未确定的抗原决定簇，在本文中，只要能结合抗原上的两种不同抗原决定簇的抗体即被称为双特异性抗体。一个具体的双特异性抗体例如可以结合PDL1和CD47。
术语“N价抗体”意在说明抗体中存在N个抗原结合位点。例如“二价抗体”指抗体存在两个抗原结合位点，“三价抗体”指抗体存在三个抗原结合位点。天然的人免疫球蛋白分子通常具有两个抗原结合位点，Fab通常具有单个抗原结合位点，单可变结构域、ScFv通常具有单个抗原结合位点。
术语“抗原结合部分”指特异性结合抗原决定簇的多肽分子。具体的抗原结合部分可以是Fab、ScFv、单可变结构域或其他结构。本文中的抗原决定簇与抗原表位同义。
本文中关于抗原结合部分、抗原、Fc多肽、肽接头、重链、轻链、多肽等所用的术语“第一”、“第二”等是为了在存在一个以上各类型的部分时方便区分而使用。除非明确说明，这些术语的使用并非旨在赋予双特异性抗体的具体顺序或取向。
术语“融合”意指组分(例如抗原结合部分、Fc多肽等)直接或经一个或多个肽接头通过肽键连接。例如一些肽接头由通过肽键连接的1到50个氨基酸组成，其中所述氨基酸可以选自20个天然存在的氨基酸；在更优选的实施方案中，1到50个氨基酸选自甘氨酸、丙氨酸、脯氨酸、丝氨酸、天冬酰胺、谷氨酰胺和赖氨酸。因此，示例性的肽接头可以是聚甘氨酸(尤其是(Gly) 4、(Gly) 5)、聚(Gly-Ser)、 (Gly) 3AsnGlySer(Gly) 2、(Gly) 3Cys(Gly) 4、GlyProAsnGlyGly，或者专利申请WO2019195535的表4所公开的那些，等等。当与Fc融合时，例如抗原结合部分与Fc融合时，典型的是通过铰链区(肽接头的其中一种形式)融合。一种实施方案，所述第一抗原结合部分经第一铰链与Fc域的其中一个Fc多肽融合，所述第二抗原结合部分经第二铰链与Fc域的另一个Fc多肽融合。第一铰链或/和第二铰链可以包含人IgG(例如IgG1、IgG4等)的铰链区的氨基酸，人IgG的铰链区包含天然的铰链区或其变体。
术语“可变结构域”或“可变区”是指抗体的涉及该抗体与抗原结合的结构域。例如天然四链抗体(例如来源于人、鼠等)具有重链可变区(VH)和轻链可变区(VL)，来源于骆驼科或鲨鱼等动物的仅重链抗体具有单可变结构域。大多数情况下，天然抗体的每个可变结构域基本上由四个“框架区”和三个“互补决定区”组成。四个框架区分别称为框架区1(FR1)、框架区2(FR2)、框架区3(FR3)、及框架区4(FR4)；所述框架区由本领域及下文中分别称为互补决定区(CDR)间隔开。因此，可变结构域的一般结构可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。一个具体的例子，重链可变区的一般结构可如下表示为：FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4。一个具体的例子，轻链可变区的一般结构可如下表示为：FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4。可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
“CDR”(互补决定区)，也称为“高变区(HVR)”。天然四链抗体通常包含六个CDR，三个在重链可变区中，分别为重链CDR1(HCDR1)、重链CDR2(HCDR2)和重链CDR3(HCDR3)，另外三个在轻链可变区中，分别为轻链CDR1(LCDR1)、轻链CDR2(LCDR2)和轻链CDR3(LCDR3)。仅重链抗体或单可变结构域通常具有三个CDR(CDR1、CDR2和CDR3)。
当前有许多方法来划分定义CDR。其中，Kabat定义基于序列可变性划分CDR，并且是最常用的(Elvin A.Kabat,et al，Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991))；而Chothia定义则基于结构环的位置(Cyrus Chothia,et al,Canonical Structures for the Hypervariable Regions of Immunoglobulins,J.Mol.Biol.196:901-917(1987))。AbM定义是Kabat定义和Chothia定义之间的折衷方案，并且被Oxford Molecular的AbM抗体建模软件使用。“接触(contact)”定义CDR的基础是对可用的复合物晶体结构的分析。然而，应当注意的是，基于不同的方法划分定义获得的同一抗体可变区的CDR的边界可能有所差异，即不同方法划分定义的同一抗体可变区的CDR序列可能有所不同。因此，在涉及用本发明划分定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了转换为其他任意定义(例如Chothia、AbM定义等)的CDR序列限定的抗体。
术语“框架区”(FR)是除了本文定义的CDR残基之外的那些可变结构域的氨基酸残基。
术语“Fab”指由免疫球蛋白的重链的VH和CH1以及轻链的VL和CL域组成的蛋白。本文中Fab意指处于它的天然型式的或经修饰的Fab，一般情况下，天然型式的Fab包含由重链可变区和恒定区CH1构成的Fab重链(VH-CH1，N至C端方向)，和由轻链可变区和恒定区CL构成的Fab轻链(VL-CL，N至C端方向)。经修饰的Fab例如可以是在CH1/CL结构域或/和VH/VL结构域引入氨基酸取代的Fab，一个具体的实例，经修饰的Fab可以是在CL结构域引入氨基酸取代的Fab。经修饰的Fab例如还可以是具有结构域替换的Fab，一个具体的例子，结构域替换为Fab中的VH/VL彼此替换或/和CH1/CL彼此替换。如无特殊说明，Fab中的Fab重链是指包含VH的多肽链，Fab轻链是指包含VL的多肽链。在一些实施方案中，轻链恒定区是人κ链恒定区。在一些实施方案中，轻链恒定区是人λ链恒定区。一些实施方案中，重链恒定区可来自任何类型的恒定区，例如IgG、IgM、IgD、IgA和IgE；以及任何同种型，例如IgG1、IgG2、IgG3和IgG4。
术语“scFv”包括免疫球蛋白的VH和VL结构域，其中这些结构域存在于单一多肽链中。在一些实施方案中，scFv还包含介于VH和VL结构域之间的肽接头，其使得scFv能够形成抗原结合所需的结构。
术语“Fc结构域”、“Fc”或“Fc域”在本文中用来定义免疫球蛋白重链的C端区域，其包含至少部分恒定区。该术语包括天然序列Fc和变体Fc。Fc的C末端赖氨酸(Lys447)可存在或不存在。除非另有说明，Fc或恒定区中氨基酸残基的编号是根据EU编号***，也称为EU索引，描述于Kabat，E.A等人，Sequences of Proteins of Immunological Interest，第5版，Public Health Service,National Institutes of Health,Bethesda,MD(1991),NIH Publication 91-3242。第一配对结构域和第二配对结构域涉及氨基酸位置的描述时，是指按照相应序列的起始氨基酸为第一位进行顺序编号。本文所用的Fc域的其中一个“Fc多肽” 指形成二聚体Fc结构域的两条多肽之一。例如，IgG Fc域的Fc多肽包含IgG CH2和IgG CH3恒定区。
术语“效应子功能”指可归因于抗体的Fc结构域的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例例如：C1q结合和依赖补体的细胞毒性(CDC)、Fc受体结合、依赖抗体的细胞毒性(ADCC)、依赖抗体的吞噬作用(ADCP)等等。
术语“K D”在用于本文时指平衡解离常数，以摩尔浓度(M)表示。抗体的K D值可以使用本领域公知的方法测定。例如，一种测定抗体K D的方法是使用表面等离子共振(surface plasmon resonance)，如使用生物传感器***，例如Biacore***。
术语“治疗”指试图改变治疗个体中疾病的自然进程，并且可以是为了预防或在临床病理学的过程期间实施的临床干预。治疗的期望效果包括但不限于预防疾病的发生或复发，缓解症状，降低疾病的任何直接或间接病理学后果，预防转移，减缓疾病进展率，改善或减轻疾病状态，及消退或改善的预后。
术语“受试者”包括任何人类或非人动物。术语“非人动物”包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、绵羊、犬、猫、马、牛、鸡、两栖动物、爬行动物等。优选地，根据本发明的受试者是人。除非标明，术语“患者”或“受试者”可以互换使用。
术语“分离的”是指目标化合物(例如，抗体或核酸等)已经从其天然环境中分离。
氨基酸序列的“同一性百分数(％)”是指将待比对序列与本文中所示的具体氨基酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，并且不以任何保守置换作为序列同一性的一部分时，待比对序列中与本文中所示的具体氨基酸序列的氨基酸残基相同的氨基酸残基百分数。氨基酸序列的同一性比对可以采用本领域范围内的多种方式进行，例如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定用于比对序列的适宜参数，包括在比较序列的全长里获得最大比对需要的任何算法。
本发明上下文中，氨基酸取代表示为：原氨基酸-位置-取代的氨基酸，使用三字母码或单字母码，包括编码Xaa和X来表示氨基酸残基。因此，例如“H435R”意为将位置435处的氨基酸H取代为氨基酸R；对于取代的氨基酸可以包含多于1个，例如T366Y/W意为将位置366处的氨基酸T取代为氨基酸Y或W。
术语“Xn”和“Xaa”等同，是指未指定的氨基酸(Unspecified Amino Acid)，通过相关表述中的后续定义来指定其涵盖的范围。
术语“包括”、“含有”或“包含”及其变体应理解为“包括但不限于”，意味着除所列出的要素、组分和步骤外，还可涵盖其它未指明的要素、组分和步骤。
在本文中，除非上下文另有明确规定，否则单数术语涵盖复数指代物，反之亦然。
为了描述和公开的目的，以引用的方式将所有的专利、专利申请和其它已确定的出版物在此明确地并入本文。这些出版物仅因为它们的公开早于本申请的申请日而提供。所有关于这些文件的日期的声明或这些文件的内容的表述是基于申请者可得的信息，并且不构成任何关于这些文件的日期或这些文件的内容的正确性的承认。而且，在任何国家，在本文中对这些出版物的任何引用并不构成关于该出版物成为本领域的公知常识的一部分的认可。
本发明的各个方面将在下述部分中进一步详细描述。
I.双特异性抗体
本发明提供了双特异性抗体，具有诸多用于靶向治疗的期望的特性。例如具有以下的一方面或多方面的特性：
(1)所述双特异性抗体可以特异性结合CD47(例如人CD47)和PD-L1(例如人PD-L1)，并具有良好的亲和力；通过结合CD47，可以阻断CD47与SIRPα的结合，促进巨噬细胞介导的对表达CD47肿瘤细胞的吞噬作用；通过结合PD-L1，可以抑制T细胞上的PD-1与肿瘤细胞表面PD-L1的结合，诱导T细胞活化从而发挥抗肿瘤作用；
(2)所述双特异性抗体不引起实质性的红细胞减少、贫血或红细胞凝集，安全性好；
(3)所述双特异性抗体具有良好的稳定性；
(4)所述双特异性抗体的分子设计易于下游工艺的纯化；
在一些实施方案中，所述双特异性抗体具有优异的抗肿瘤作用。一些实验表明，所述双特异性抗体对 肿瘤例如人淋巴瘤、黑色素瘤等具有优异的抗肿瘤作用。
本发明提供了一种双特异性抗体，其包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含：
重链可变区，其包含：
HCDR1，其包含SEQ ID NO：1、2、3、4或5所示的氨基酸序列，
HCDR2，其包含SEQ ID NO：6、7、8、9或10所示的氨基酸序列，和
HCDR3，其包含SEQ ID NO：11、12、13、14或15所示的氨基酸序列；和
轻链可变区，其包含：
LCDR1，其包含SEQ ID NO：16、17、18、19或20所示的氨基酸序列，
LCDR2，其包含SEQ ID NO：21、22、23、24或25所示的氨基酸序列，和
LCDR3，其包含SEQ ID NO：26、27、28、30或32所示的氨基酸序列。
在一些实施方案中，所述第一抗原结合部分包含：
重链可变区，其包含：包含SEQ ID NO：1所示氨基酸序列的HCDR1，包含SEQ ID NO：6所示氨基酸序列的HCDR2，和包含SEQ ID NO：11所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：16所示氨基酸序列的LCDR1，包含SEQ ID NO：21所示氨基酸序列的LCDR2，和包含SEQ ID NO：26所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分包含：
重链可变区，其包含：包含SEQ ID NO：2所示氨基酸序列的HCDR1，包含SEQ ID NO：7所示氨基酸序列的HCDR2，和包含SEQ ID NO：12所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：17所示氨基酸序列的LCDR1，包含SEQ ID NO：22所示氨基酸序列的LCDR2，和包含SEQ ID NO：27所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分包含：
重链可变区，其包含：包含SEQ ID NO：3所示氨基酸序列的HCDR1，包含SEQ ID NO：8所示氨基酸序列的HCDR2，和包含SEQ ID NO：13所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：18所示氨基酸序列的LCDR1，包含SEQ ID NO：23所示氨基酸序列的LCDR2，和包含SEQ ID NO：28所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分包含：
重链可变区，其包含：包含SEQ ID NO：4所示氨基酸序列的HCDR1，包含SEQ ID NO：9所示氨基酸序列的HCDR2，和包含SEQ ID NO：14所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：19所示氨基酸序列的LCDR1，包含SEQ ID NO：24所示氨基酸序列的LCDR2，和包含SEQ ID NO：32所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分包含：
重链可变区，其包含：包含SEQ ID NO：5所示氨基酸序列的HCDR1，包含SEQ ID NO：10所示氨基酸序列的HCDR2，和包含SEQ ID NO：15所示氨基酸序列的HCDR3；和
轻链可变区，其包含：包含SEQ ID NO：20所示氨基酸序列的LCDR1，包含SEQ ID NO：25所示氨基酸序列的LCDR2，和包含SEQ ID NO：30所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO：33、35、37、39、41、83、85或87所示可变区序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含SEQ ID NO：34、36、38、40、42、84、86或88所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：33所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：34所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：35所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：36所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：37所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结 合部分的轻链可变区包含SEQ ID NO：38所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：39所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：40所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：41所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：42所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：83所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：84所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：85所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：86所示可变区序列的LCDR1、LCDR2和LCDR3。在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：87所示可变区序列的HCDR1、HCDR2和HCDR3，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：88所示可变区序列的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：33、35、37、39、41、83、85或87所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。
在一些实施方案中，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：34、36、38、40、42、84、86或88所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：33所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：34所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：1所示氨基酸序列的HCDR1、包含SEQ ID NO：6所示氨基酸序列的HCDR2、包含SEQ ID NO：11所示氨基酸序列的HCDR3、包含SEQ ID NO：16所示氨基酸序列的LCDR1、包含SEQ ID NO：21所示氨基酸序列的LCDR2和包含SEQ ID NO：26所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：35所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：36所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：3所示氨基酸序列的HCDR1、包含SEQ ID NO：8所示氨基酸序列的HCDR2、包含SEQ ID NO：13所示氨基酸序列的HCDR3、包含SEQ ID NO：18所示氨基酸序列的LCDR1、包含SEQ ID NO：23所示氨基酸序列的LCDR2和包含SEQ ID NO：28所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：37所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：38所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：2所示氨基酸序列的HCDR1、包含SEQ ID NO：7所示氨基酸序列的HCDR2、包含SEQ ID NO：12所示氨基酸序列的HCDR3、包含SEQ ID NO：17所示氨基酸序列的LCDR1、包含SEQ ID NO：22所示氨基酸序列的LCDR2，和包含SEQ ID NO：27所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：39所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％ 的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：40所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：4所示氨基酸序列的HCDR1、包含SEQ ID NO：9所示氨基酸序列的HCDR2、包含SEQ ID NO：14所示氨基酸序列的HCDR3、包含SEQ ID NO：19所示氨基酸序列的LCDR1、包含SEQ ID NO：24所示氨基酸序列的LCDR2和包含SEQ ID NO：32所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：41所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：42所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：5所示氨基酸序列的HCDR1、包含SEQ ID NO：10所示氨基酸序列的HCDR2、包含SEQ ID NO：15所示氨基酸序列的HCDR3、包含SEQ ID NO：20所示氨基酸序列的LCDR1、包含SEQ ID NO：25所示氨基酸序列的LCDR2和包含SEQ ID NO：30所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：83所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：84所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：4所示氨基酸序列的HCDR1、包含SEQ ID NO：9所示氨基酸序列的HCDR2、包含SEQ ID NO：14所示氨基酸序列的HCDR3、包含SEQ ID NO：19所示氨基酸序列的LCDR1、包含SEQ ID NO：24所示氨基酸序列的LCDR2和包含SEQ ID NO：32所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：85所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：86所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：3所示氨基酸序列的HCDR1、包含SEQ ID NO：8所示氨基酸序列的HCDR2、包含SEQ ID NO：13所示氨基酸序列的HCDR3、包含SEQ ID NO：18所示氨基酸序列的LCDR1、包含SEQ ID NO：23所示氨基酸序列的LCDR2和包含SEQ ID NO：28所示氨基酸序列的LCDR3。
在一些实施方案中，所述第一抗原结合部分的重链可变区包含与SEQ ID NO：87所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：88所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。进一步的，在一些这种实施方案中，所述第一抗原结合部分还包含包含SEQ ID NO：5所示氨基酸序列的HCDR1、包含SEQ ID NO：10所示氨基酸序列的HCDR2、包含SEQ ID NO：15所示氨基酸序列的HCDR3、包含SEQ ID NO：20所示氨基酸序列的LCDR1、包含SEQ ID NO：25所示氨基酸序列的LCDR2和包含SEQ ID NO：30所示氨基酸序列的LCDR3。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：33所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：34所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：35所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：36所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：37所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：38所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：39所示的氨基酸序 列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：40所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：41所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：42所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：83所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：84所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：85所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：86所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：87所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：88所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：81所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：82所示的氨基酸序列。
在一些具体的实施方案中，所述第一抗原结合部分的重链可变区包含SEQ ID NO：79所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：80所示的氨基酸序列。
在另一些具体的实施方案中，所述的第一抗原结合部分的重链可变区和轻链可变区包含选自SEQ ID NO：43/44、45/46、47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70、71/72、73/74、75/76、77/78所示的氨基酸序列。
在一些实施方案中，所述的第一抗原结合部分是鼠源的或人源化的。人源化可降低免疫原性，一个实例，所述的第一抗原结合部分是人源化的。
表S1和表S2提供了一些示例性的结合CD47的单抗例如2B2、2H8、3F10、16E5和14A9抗体的嵌合版本(Xi2B2、Xi2H8、Xi3F10、Xi16E5和Xi14A9)以及3F10、16E5、14A9的人源化变体的CDR序列。表S3提供了这些示例性的单抗的可变区序列。这些示例性的抗体，可以用于构建本文所述的双特异性抗体。例如，一些实施方案中，所述的双特异性抗体的第一抗原结合部分的CDR选自任一示例性的结合CD47的单抗的六个CDR。例如，一些实施方案中，所述双特异性抗体的第一抗原结合部分的可变区选自任一示例性的结合CD47的单抗的重链可变区和轻链可变区。
表S1：重链CDR序列
Figure PCTCN2022122531-appb-000001
表S2：轻链CDR序列
Figure PCTCN2022122531-appb-000002
表S3：可变区序列
Figure PCTCN2022122531-appb-000003
Figure PCTCN2022122531-appb-000004
Figure PCTCN2022122531-appb-000005
Figure PCTCN2022122531-appb-000006
Figure PCTCN2022122531-appb-000007
Figure PCTCN2022122531-appb-000008
所述第二抗原结合部分提供结合PD-L1的能力，例如可以结合灵长类如人的PD-L1。在一些实施方案中，所述第二抗原结合部分是鼠源的、人源化的或全人源的。一个实例，所述第一抗原结合部分和第二抗原结合部分均为人源化的；另一个实例，所述第一抗原结合部分是人源化的，所述第二抗原结合部分是全人源的。
在一些实施方案中，所述第二抗原结合部分包含重链可变区和轻链可变区，其中重链可变区包含：包含SEQ ID NO：199所示氨基酸序列的HCDR1，包含SEQ ID NO：200所示氨基酸序列的HCDR2，和包含SEQ ID NO：201所示氨基酸序列的HCDR3；轻链可变区包含：包含SEQ ID NO：202所示氨基酸序列的LCDR1，包含SEQ ID NO：203所示氨基酸序列的LCDR2，和包含SEQ ID NO：204所示氨基酸序列的LCDR3。
在一些实施方案中，其中所述第二抗原结合部分的重链可变区包含SEQ ID NO：205所示的重链可变区序列的HCDR1，HCDR2和HCDR3；所述第二抗原结合部分的轻链可变区包含SEQ ID NO：206所示的轻链可变区序列的LCDR1，HCDR2和LCDR3。
在一些实施方案中，所述第二抗原结合部分的重链可变区包含与SEQ ID NO：205所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列，所述第二抗原结合部分的轻链可变区包含与SEQ ID NO：206所示序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。一个更具体的方案，第二抗原结合部分中，所述的重链可变区包含SEQ ID NO：205所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：206所示的氨基酸序列。
表S4示例性的第二抗原结合部分的CDR和可变区序列。
表S4：示例性的第二抗原结合部分的CDR和可变区序列
Figure PCTCN2022122531-appb-000009
多特异性抗体构型
在一些实施方案中，本文的所述双特异性抗体可以是无Fc域的，所述第一抗原结合部分、第二抗原结合部分通过合适的接头融合。
在另一些实施方案中，本文的所述双特异性抗体可以是有Fc域的。一些情况，Fc域可以延长半衰期、提供Fc域相关的效应子等功能。所述第一抗原结合部分或/和第二抗原结合部分可以与Fc域的N端或C端融合。
所述第一抗原结合部分和第二抗原结合部分可以采用任意适宜的结构形式。在一些实施方案中，所述的第一抗原结合部分和第二抗原结合部分可以各自独立的为Fab、ScFv、ScFab或其他结构。
所述的双特异性抗体是多价的，例如二价、四价等。在一些实施方案中，所述的双特异性抗体是二价的，即第一抗原结合部分、第二抗原结合部分均各自提供与相应抗原的单价结合。
在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分均为Fab。
在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分的其中之一具有结构域替换，所述结构域替换为
重链可变区和轻链可变区彼此替换，或/和
恒定结构区CH1和CL彼此替换。这类结构域替换防止来自不同Fab的重链和轻链的错配，从而提高重组产生中双特异性抗体的产率和纯度。换言之，通过双特异性抗体的第一抗原结合部分或第二抗原结合部分内的可变区和/或恒定区的交换来克服双特异性抗体产生中重链和轻链错配的问题，使得不同特异性的Fab不具有相同的结构域排列，并因此而不“交换”轻链。
在一些实施方案中，所述结构域替换为重链可变区和轻链可变区替换，使得抗原结合部分包含由轻链可变区和重链恒定区构成的肽链和由重链可变区和轻链恒定区构成的肽链。在一些实施方案中，所述的结构域替换为恒定结构区CH1和CL彼此替换，使得抗原结合部分包含由重链可变区和轻链恒定区构成的肽链和由轻链可变区和重链恒定区构成的肽链。在一些实施方案中，所述的结构域替换为重链可变区和轻链可变区彼此替换，以及恒定结构区CH1和CL彼此替换。
为了达到希望的防止不同特异性的重链和轻链的错配，必须在不同特异性的抗原结合部分中进行不一样的结构域替换或者结构域替换仅发生在同一抗原结合部分。例如，在第一抗原结合部分中可以交换重链可变区和轻链可变区，而在第二抗原结合部分中，可以交换恒定结构区CH1和CL。例如，在第一抗原结合部分中，可以不进行结构域替换，而在第二抗原结合部分中，可以交换恒定结构区CH1和CL。
在一些实施方案中，所述双特异性抗体还包含由两个Fc多肽构成的Fc域。
在一些实施方案中，所述第一抗原结合部分在其C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分在其C端融合至所述Fc域的另一个Fc多肽的N端。
在一些实施方案中，所述第一抗原结合部分为Fab且所述第一抗原结合部分在其Fab重链的C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分为Fab且所述第二抗原结合部分在其Fab重链的C端融合至所述Fc域的另一个Fc多肽的N端。
在一些具体的实施方案中，所述第一抗原结合部分为Fab且所述第一抗原结合部分在其Fab重链的C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分为Fab且所述第二抗原结合部分在其Fab重链的C端融合至所述Fc域的另一个Fc多肽的N端，其中第二抗原结合部分中恒定结构区CH1和CL彼此替换。一种具体的构型如图1所示。
Fc域
双特异性抗体的Fc域由一对包含免疫球蛋白分子重链结构域的多肽链组成。例如，免疫球蛋白G(IgG)分子的Fc域是二聚体，其每个Fc多肽包含CH2和CH3IgG重链恒定区。Fc域的两个Fc多肽能够彼此稳定缔合。在一个实施方案中，所述的双特异性抗体包含一个Fc域。
在一个实施方案中，多特异性抗体的Fc域是IgG Fc域。在一个具体的实施方案中，Fc域是IgG1Fc域。在另一个实施方案中，Fc域是IgG4Fc域。在一个更具体的实施方案中，Fc域是包含位置S228处的氨基酸替代，特别是氨基酸替代S228P的IgG4Fc域，此氨基酸替代降低IgG4抗体的体内Fab臂交换。在又一个具体的实施方案中，Fc域是人Fc域。在一个更具体的实施方案中，Fc域是人IgG1Fc域。
在一些实施方案中，所述Fc域包含修饰，例如氨基酸取代。所述修饰例如可以是促进异二聚化的修饰，改变效应子功能的修饰或改变与A蛋白结合能力的修饰等。
在一些实施方案中，所述Fc包含促进异二聚化的修饰。
在某些情况下，所述的双特异性抗体包含与Fc域中的两个Fc多肽中的一个或另一个融合的不同抗原结合部分，因此，两个Fc多肽通常包含在两条不同的多肽链中。这些多肽的重组共表达和随后的二聚化产生两种多肽的几种可能的组合。为了提高重组产生中多特异性抗体的产率和纯度，在该双特异性抗体的Fc域中引入促进希望的多肽结合的修饰将是有利的。因此，在一些具体实施方案中，所述Fc域包含促进所述Fc域的两个Fc多肽相缔合的氨基酸取代。
人IgG Fc域的两条Fc多肽之间最广泛的蛋白质-蛋白质相互作用的部位在Fc域的CH3结构域中。因此，在一个实施方案中，该氨基酸取代在Fc域的CH3结构域中。
在具体实施方案中，该氨基酸取代是所谓的“杵入臼(knob-into-hole)”，其包含Fc域的两个Fc多肽之一中的“杵”氨基酸取代和Fc域的两个Fc多肽的另一个中的“臼”氨基酸取代。通常，该方法涉及在一条Fc多肽的界面引入凸起(“杵”)，而在另一条Fc多肽的界面引入对应的凹陷(“臼”)，使得该凸起可以定位在该凹陷中，以促进异二聚体形成和妨碍同二聚体形成。通过用更大的侧链(例如酪氨酸或色氨酸等)取代来自一条Fc多肽的界面的小的氨基酸侧链来构建凸起。通过用更小的氨基酸侧链(例如丙氨酸或苏氨酸等)取代大的氨基酸侧链来在另一条Fc多肽的界面中产生具有与凸起相同或相似的大小的互补凹陷。
因此，在一些具体实施方案中，在该双特异性抗体的一个Fc多肽的CH3结构域中，用具有更大侧链体积的氨基酸残基取代氨基酸残基，从而在该Fc多肽的CH3结构域内产生可定位在另一个Fc多肽的CH3结构域内的凹陷中的凸起，在另一个Fc多肽的CH3结构域中，用具有更小侧链体积的氨基酸残基取代氨基酸残基，从而在该Fc多肽的CH3结构域内产生凹陷。
在一些具体实施方案中，所述Fc域的其中一个Fc多肽包含T366Y/W或/和S354C，且另一个Fc多肽包含Y407T/V、Y349C、T366S或/和L368A。一个更具体的实例，所述Fc域的其中一个Fc多肽包含氨基酸取代T366Y/W和S354C，另一个Fc多肽包含氨基酸取代Y407T/V、Y349C、T366S和L368A。在更具体的实例中，Fc可以是人IgG1的Fc。
在一些实施方案中，所述Fc域包含改变效应子功能的修饰。在一些具体的实施方案中，将该双特异性抗体的Fc域进行修饰为与未进行所述修饰的Fc相比，降低Fc域对Fc受体的结合亲和力和/或效应子功能。降低Fc域对Fc受体的结合亲和力和/或效应子功能对改善细胞因子释放或/和副作用是有利的。
在一些实施方案中，降低该Fc域与Fc受体的结合亲和力和/或减少效应子功能的修饰是氨基酸取代。在一些实施方案中，所述Fc域在选自E233、L234、L235、N297、P331和P329的一个或多个位置上包含氨基酸取代。在一些实施方案中，所述Fc域在选自L234、L235和P329的一个或多个位置上包含氨基酸取代。在一些实施方案中，所述Fc域包含氨基酸取代L234A和L235A。在一个这种实施方案中，所述Fc域是IgG1Fc，尤其是人IgG1Fc。在一些实施方案中，所述Fc域在P329位包含氨基酸取代。在一些更具体的实施方案中，该氨基酸取代是P329A、P329R或P329G。在一些实施方案中，该Fc域包含P329位的氨基酸取代及选自E233、L234、L235、N297和P331的位置上的另一氨基酸取代。在更具体的实施方案中，该另一氨基酸取代是E233P、L234A、L235A、L235E、N297A、N297D或P331S。在一些实施方案中，该Fc域在P329、L234和L235位包含氨基酸取代。在一些更具体的实施方案中，该Fc域包含氨基酸取代L234A、L235A和P329G(“P329G LALA”)。在更具体的另一个实施方案中，该Fc域包含氨基酸取代L234A、L235A和P329A(“P329A LALA”)。在一个这种实施方案中，该Fc是IgG1Fc，尤其是人IgG1Fc。
在一些具体实施方案中，所述Fc域是包含氨基酸取代L234A、L235A和P329A/G/R的人IgG1Fc结构域。在一些具体实施方案中，所述Fc域是包含氨基酸取代L234A、L235A的人IgG1Fc结构域。
以上所述降低该Fc域与Fc受体的结合亲和力和/或效应子功能的氨基酸取代发生在Fc域的两条多肽链上。
在一些实施方案中，所述Fc域包含减少或消除Fc域中的一个Fc多肽的CH3区与A蛋白(Protein A from Staphylococcus aureus)的结合的修饰。在一些实施方案中，该修饰为氨基酸取代。在一些实施方案中，所述Fc域包含氨基酸取代H435R或/和Y436F，该氨基酸取代仅发生在一个Fc多肽上，而不发生在另一个Fc多肽上。在一个具体的实施方案中，所述Fc域包含仅发生在其中一个Fc多肽的氨基酸取代H435R或/和Y436F。在一个这种具体的实施方案中，该Fc是IgG1Fc，尤其是人IgG1Fc。
在一些实施方案中，所述的双特异性抗体中，所述Fc域可以包含促进异二聚化的修饰、改变效应子功能的修饰、减少或消除Fc中的一条Fc多肽的CH3区与A蛋白的结合的修饰中的一种、两种或三种。在一些实施方案中，所述Fc域包含促进两个Fc多肽缔和的修饰、所述降低Fc域对Fc受体的结合亲和力和/或减少的效应子功能的修饰、以及减少或消除Fc中的一个Fc多肽的CH3区与A蛋白的结合的修饰。对于上述不同种修饰类型的具体方案，可以进行组合。
在一些实施方案中，所述双特异性抗体包含：
第一重链，其包含所述第一抗原结合部分的重链可变区；
第一轻链，其包含所述第一抗原结合部分的轻链可变区；
第二重链，其包含所述第二抗原结合部分的重链可变区；和
第二轻链，其包含所述第二抗原结合部分的轻链可变区。
一些示例性的方案，所述第一重链包含与SEQ ID NO：207所示序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，所述第一轻链包含SEQ ID NO：208所示序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，所述第二重链包含与SEQ ID NO：209所示序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，所述第二轻链包含SEQ ID NO：210所示序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
一些具体的实施方案，所述第一重链包含SEQ ID NO：207所示的氨基酸序列，所述第一轻链包含SEQ ID NO：208所示的氨基酸序列，所述第二重链包含SEQ ID NO：209所示的氨基酸序列，所述第二轻链包含SEQ ID NO：210所示的氨基酸序列。
在一些实施方案中，所述双特异性抗体以1E-08M或更小的K D值与CD47(例如人CD47)结合。在一些实施方案中，所述双特异性抗体对人CD47具有下述结合亲和力(K D)：在约1E-11M至约1E-08M、约1E-10M至约1E-08M、约5E-09M至约1E-08M的范围内。在一些实施方案中，所述双特异性抗体对人CD47具有下述结合亲和力(K D)：约1E-08M或更小、约5E-09M或更小、约3E-09M或更小、约1E-09M或更小、或者约1E-10M或更小。在一些实施方案中，通过Biacore来测量本文双特异性抗体与CD47的结合亲和力K D。
在一些实施方案中，所述双特异性抗体以1E-09M或更小的K D值与PD-L1(例如人PD-L1)结合。在一些实施方案中，所述双特异性抗体对人PD-L1具有下述结合亲和力(K D)：在约1E-11M至约1E-08M、约1E-11M至约1E-09M、约5E-10M至约1E-10M的范围内。在一些实施方案中，所述双特异性抗体对人PD-L1具有下述结合亲和力(K D)：约1E-09M或更小、约1E-10M或更小、约5E-10M或更小、或者约1E-11M或更小。在一些实施方案中，通过Biacore来测量本文双特异性抗体与PD-L1的结合亲和力K D。
II.双特异性抗体的变体
在一些实施方案中，提供双特异性抗体的变体。可以通过向编码双特异性抗体的核苷酸序列引入适当修饰或通过肽合成制备双特异性抗体的氨基酸序列变体。此类修饰，例如，从抗体的氨基酸序列内部缺失氨基酸残基，将氨基酸残基***所述氨基酸序列，或/和置换所述氨基酸序列中的氨基酸残基。可以产生缺失、***和置换的任意组合以获得最终构建体，只要所述最终变体拥有想要的特征，例如抗原结合作用，例如肿瘤杀伤效能等。
III.药物组合物
本发明提供了药物组合物，所述药物组合物包含双特异性抗体，并且还包含一种或多种药学上可接受的载体。药学上可接受的载体包括，例如，赋形剂、稀释剂、包封材料、填充剂、缓冲剂或其他试剂。
所述的药物组合物适于多种给药方式，一个例子为静脉内施用。
Ⅳ.分离的核酸
本发明提供了分离的核酸，其包含编码本发明所述的双特异性抗体。序列表中示例性的列举了一些双特异性抗体的核酸序列。
Ⅴ.载体
本发明提供了包含所述的核酸的载体。在一些实施方案中，所述的载体为克隆载体；在另一些实施方案中，所述的载体为表达载体，一个具体的例子，表达载体为pcDNA3.1(+)。所述表达载体可选的能够表达本文所述双特异性抗体的任意表达载体。
Ⅵ.宿主细胞
在一些实施方案中，本发明提供包含所述载体的宿主细胞，宿主细胞为用于克隆或表达双特异性抗体 的适当宿主细胞。在一些实施方案中，宿主细胞为原核细胞。在另一些实施方案中，宿主细胞为真核细胞。在一些实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备双特异性抗体的其他细胞。哺乳动物细胞例如为中国仓鼠(CHO)卵巢细胞、CHO-S细胞等。
Ⅶ.制备双特异性抗体的方法
在一些实施方案中，本发明提供了制备双特异性抗体的方法，所述方法包括：培养所述的宿主细胞，使其表达所述的双特异性抗体，并且在体系中分离纯化所述双特异性抗体。为了产生所述的双特异性抗体，分离编码所述双特异性抗体的核酸，并***一个或多个载体，用于在宿主细胞中进一步克隆或/和表达。所述核酸可以采用基因拼接、化学合成等多种本领域所熟知的方法获取。
所制备的双特异性抗体可以通过已知的现有技术例如亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于如净电荷、疏水性等因素，并且这些对于本领域技术人员是显而易见的。
可以通过多种熟知的分析方法中的任何一种确定双特异性抗体的纯度，例如大小排阻层析等等。可以通过本领域已知的多种测定方法鉴定、筛选或表征本文的双特异性抗体的理化性质或/和生物学活性。
Ⅷ.用途
本发明提供了一种用于在受试者中减少或抑制肿瘤细胞生长的方法，所述方法包括向所述的受试者施用治疗有效量的所述双特异性抗体。
本发明提供了所述的双特异性抗体或所述的药物组合物在制备治疗癌症的药物中的用途。
本发明提供了在受试者中治疗癌症的方法，其包括对所述受试者施用治疗有效量的所述的双特异性抗体或所述的药物组合物。
其中所述癌症为白血病、淋巴瘤、骨髓瘤、卵巢癌、乳腺癌、子宫内膜癌、结直肠癌、结肠癌、直肠癌、膀胱癌、尿路上皮癌、肺癌、支气管癌、骨癌、***癌、胰腺癌、胃癌、肝癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、***、以及子宫癌、成胶质细胞瘤、成神经管细胞瘤、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、骨髓增生异常综合征、神经胶质瘤、唾液腺癌、胸腺癌、上皮癌、胃食管交接部癌、食管癌、胸膜间皮瘤、鼻咽癌或肉瘤。
本发明提供了一种用于增加T细胞活化的方法，所述方法包括使T细胞与所述的双特异性抗体接触。
本发明提供了一种促进受试者巨噬细胞的吞噬作用的方法，所述方法包括向所述的受试者施用治疗有效量的所述双特异性抗体。
本发明还提供了以下一些具体的实施方案，但本发明的保护范围不限于此：
实施方案1.一种双特异性抗体，其包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含：
重链可变区，其包含：
HCDR1，其包含SEQ ID NO：5、1、2、3或4所示的氨基酸序列，
HCDR2，其包含SEQ ID NO：10、6、7、8或9所示的氨基酸序列，和
HCDR3，其包含SEQ ID NO：15、11、12、13或14所示的氨基酸序列；和
轻链可变区，其包含：
LCDR1，其包含SEQ ID NO：20、16、17、18或19所示的氨基酸序列，
LCDR2，其包含SEQ ID NO：25、21、22、23或24所示的氨基酸序列，和
LCDR3，其包含SEQ ID NO：30、26、27、28或32所示的氨基酸序列。
实施方案2.根据实施方案1所述的双特异性抗体，其中所述第一抗原结合部分包含：
(v)重链可变区，其包含：
包含SEQ ID NO：5所示氨基酸序列的HCDR1，
包含SEQ ID NO：10所示氨基酸序列的HCDR2，和
包含SEQ ID NO：15所示氨基酸序列的HCDR3；和
轻链可变区，其包含：
包含SEQ ID NO：20所示氨基酸序列的LCDR1，
包含SEQ ID NO：25所示氨基酸序列的LCDR2，和
包含SEQ ID NO：30所示氨基酸序列的LCDR3；
(i)重链可变区，其包含：
包含SEQ ID NO：1所示氨基酸序列的HCDR1，
包含SEQ ID NO：6所示氨基酸序列的HCDR2，和
包含SEQ ID NO：11所示氨基酸序列的HCDR3；和
轻链可变区，其包含：
包含SEQ ID NO：16所示氨基酸序列的LCDR1，
包含SEQ ID NO：21所示氨基酸序列的LCDR2，和
包含SEQ ID NO：26所示氨基酸序列的LCDR3；
(ii)重链可变区，其包含：
包含SEQ ID NO：2所示氨基酸序列的HCDR1，
包含SEQ ID NO：7所示氨基酸序列的HCDR2，和
包含SEQ ID NO：12所示氨基酸序列的HCDR3；和
轻链可变区，其包含：
包含SEQ ID NO：17所示氨基酸序列的LCDR1，
包含SEQ ID NO：22所示氨基酸序列的LCDR2，和
包含SEQ ID NO：27所示氨基酸序列的LCDR3；
(iii)重链可变区，其包含：
包含SEQ ID NO：3所示氨基酸序列的HCDR1，
包含SEQ ID NO：8所示氨基酸序列的HCDR2，和
包含SEQ ID NO：13所示氨基酸序列的HCDR3；和
轻链可变区，其包含：
包含SEQ ID NO：18所示氨基酸序列的LCDR1，
包含SEQ ID NO：23所示氨基酸序列的LCDR2，和
包含SEQ ID NO：28所示氨基酸序列的LCDR3；或者
(iv)重链可变区，其包含：
包含SEQ ID NO：4所示氨基酸序列的HCDR1，
包含SEQ ID NO：9所示氨基酸序列的HCDR2，和
包含SEQ ID NO：14所示氨基酸序列的HCDR3；和
轻链可变区，其包含：
包含SEQ ID NO：19所示氨基酸序列的LCDR1，
包含SEQ ID NO：24所示氨基酸序列的LCDR2，和
包含SEQ ID NO：32所示氨基酸序列的LCDR3。
实施方案3.一种双特异性抗体，其包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO：87、33、35、37、39、41、83或85所示可变区序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含SEQ ID NO：88、34、36、38、40、42、84或86所示可变区序列的LCDR1、LCDR2和LCDR3。
实施方案4.根据实施方案1-3中任一项所述的双特异性抗体，其中所述第一抗原结合部分的重链可变区包含与SEQ ID NO：33、35、37、39、41、83、85或87所示序列具有至少80％同一性的氨基酸序列。
实施方案5.根据实施方案1-4中任一项所述的双特异性抗体，其中所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：34、36、38、40、42、84、86或88所示序列具有至少80％同一性的氨基酸序列。
实施方案6.根据实施方案1-5中任一项所述的双特异性抗体，其中所述第一抗原结合部分的重链可变区包含SEQ ID NO：33、35、37、39、41、83、85或87所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：34、36、38、40、42、84、86或88所示的氨基酸序列。
实施方案7.根据实施方案1-6中任一项所述的双特异性抗体，其中所述第一抗原结合部分的重链可变区和轻链可变区选自以下任一项：包含：
(a)所述的重链可变区包含与SEQ ID NO：33所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：34所示序列具有至少80％同一性的氨基酸序列；
(b)所述的重链可变区包含与SEQ ID NO：35所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：36所示序列具有至少80％同一性的氨基酸序列；
(c)所述的重链可变区包含与SEQ ID NO：37所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：38所示序列具有至少80％同一性的氨基酸序列；
(d)所述的重链可变区包含与SEQ ID NO：39所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：40所示序列具有至少80％同一性的氨基酸序列；
(e)所述的重链可变区包含与SEQ ID NO：41所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：42所示序列具有至少80％同一性的氨基酸序列；
(f)所述的重链可变区包含与SEQ ID NO：83所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：84所示序列具有至少80％同一性的氨基酸序列；
(g)所述的重链可变区包含与SEQ ID NO：85所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：86所示序列具有至少80％同一性的氨基酸序列；以及
(h)所述的重链可变区包含与SEQ ID NO：87所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：88所示序列具有至少80％同一性的氨基酸序列。
实施方案8.根据实施方案7所述的双特异性抗体，其中所述第一抗原结合部分的重链可变区和轻链可变区选自以下任一项：
(a)所述的重链可变区包含SEQ ID NO：33所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：34所示的氨基酸序列；
(b)所述的重链可变区包含SEQ ID NO：35所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：36所示的氨基酸序列；
(c)所述的重链可变区包含SEQ ID NO：37所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：38所示的氨基酸序列；
(d)所述的重链可变区包含SEQ ID NO：39所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：40所示的氨基酸序列；
(e)所述的重链可变区包含SEQ ID NO：41所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：42所示的氨基酸序列；
(f)所述的重链可变区包含SEQ ID NO：83所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：84所示的氨基酸序列；
(g)所述的重链可变区包含SEQ ID NO：85所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：86所示的氨基酸序列；
(h)所述的重链可变区包含SEQ ID NO：87所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：88所示的氨基酸序列；
(i)所述的重链可变区包含SEQ ID NO：81所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：82所示的氨基酸序列；或者
(j)所述的重链可变区包含SEQ ID NO：79所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：80所示的氨基酸序列。
实施方案9.根据实施方案1-8中任一项所述的双特异性抗体，其中所述第二抗原结合部分包含重链可变区和轻链可变区，其中重链可变区包含：包含SEQ ID NO：199所示氨基酸序列的HCDR1，包含SEQ ID NO：200所示氨基酸序列的HCDR2，和包含SEQ ID NO：201所示氨基酸序列的HCDR3；轻链可变区包含：包含SEQ ID NO：202所示氨基酸序列的LCDR1，包含SEQ ID NO：203所示氨基酸序列的LCDR2，和包含SEQ ID NO：204所示氨基酸序列的LCDR3。
实施方案10.根据实施方案1-8中任一项所述的双特异性抗体，其中所述第二抗原结合部分的重链可变区包含SEQ ID NO：205所示的重链可变区序列的HCDR1，HCDR2和HCDR3；所述第二抗原结合部分的轻链可变区包含SEQ ID NO：206所示的轻链可变区序列的LCDR1，HCDR2和LCDR3。
实施方案11.根据实施方案9或10所述的双特异性抗体，其中所述第二抗原结合部分的重链可变区包含与SEQ ID NO：205所示序列具有至少85％同一性的氨基酸序列，所述第二抗原结合部分的轻链可变区包含与SEQ ID NO：206所示序列具有至少80％同一性的氨基酸序列。
实施方案12.根据实施方案11所述的双特异性抗体，其中所述的重链可变区包含SEQ ID NO：205所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：206所示的氨基酸序列。
实施方案13.根据实施方案1-12中任一项所述的双特异性抗体，其中所述第一抗原结合部分和第二抗原结合部分各自独立的为Fab、ScFv或ScFab。
实施方案14.根据实施方案13所述的双特异性抗体，其中所述第一抗原结合部分和第二抗原结合部分均为Fab。
实施方案15.根据实施方案14所述的双特异性抗体，其中所述第一抗原结合部分和第二抗原结合部分的其中之一具有结构域替换，所述结构域替换为
重链可变区和轻链可变区彼此替换，或/和
恒定结构区CH1和CL彼此替换。
实施方案16.根据实施方案15所述的双特异性抗体，其中所述第二抗原结合部分中，恒定结构区CH1和CL彼此替换，使得第二抗原结合部分包含由重链可变区和轻链恒定区构成的肽链和由轻链可变区和重链恒定区构成的肽链。
实施方案17.根据实施方案1-16中任一项所述的双特异性抗体，其中双特异性抗体还包含由两个Fc多肽构成的Fc域。
实施方案18.根据实施方案17所述的双特异性抗体，其中所述第一抗原结合部分在其C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分在其C端融合至所述Fc域的另一个Fc多肽的N端。
实施方案19.根据实施方案18所述的双特异性抗体，其中所述第一抗原结合部分为Fab且所述第一抗原结合部分在其Fab重链的C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分为Fab且所述第二抗原结合部分在其Fab重链的C端融合至所述Fc域的另一个Fc多肽的N端。
实施方案20.根据实施方案17-19中任一项所述的双特异性抗体，其中所述Fc域是IgG Fc域。
实施方案21.根据实施方案20所述的双特异性抗体，其中所述IgG Fc域是人IgG Fc域，优选为人IgG1或人IgG4的Fc域。
实施方案22.根据实施方案17-21中任一项所述的双特异性抗体，其中所述Fc域包含促进所述Fc域的两个Fc多肽相缔合的氨基酸取代。
实施方案23.根据实施方案17-22中任一项所述的双特异性抗体，其中所述Fc域包含降低Fc域对Fc受体的结合亲和力和/或效应子功能的氨基酸取代。
实施方案24.根据实施方案23所述的双特异性抗体，其中根据EU编号，降低Fc域对Fc受体的结合亲和力和/或效应子功能的氨基酸取代在选自下组的一个或多个位置处：L234，L235和P329。
实施方案25.根据实施方案17-24中任一项所述的双特异性抗体，其中所述Fc域包含减少或消除Fc域中的一个Fc多肽的CH3区与A蛋白的结合的氨基酸取代。
实施方案26.根据实施方案1-25中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
第一重链，其包含所述第一抗原结合部分的重链可变区；
第一轻链，其包含所述第一抗原结合部分的轻链可变区；
第二重链，其包含所述第二抗原结合部分的重链可变区；和
第二轻链，其包含所述第二抗原结合部分的轻链可变区。
实施方案27.根据实施方案26所述的双特异性抗体，其中所述第一重链包含与SEQ ID NO：207所示序列具有至少85％同一性的氨基酸序列，所述第一轻链包含与SEQ ID NO：208所示序列具有至少85％同 一性的氨基酸序列，所述第二重链包含与SEQ ID NO：209所示序列具有至少85％同一性的氨基酸序列，所述第二轻链包含与SEQ ID NO：210所示序列具有至少85％同一性的氨基酸序列。
实施方案28.根据实施方案1-27中任一项所述的双特异性抗体，其为二价的。
实施方案29.根据实施方案1-28中任一项所述的双特异性抗体，其中所述双特异性抗体是嵌合的或人源化的。
实施方案30.根据实施方案1-29中任一项所述的双特异性抗体，其中所述双特异性抗体结合人CD47的K D≤1E-08M。
实施方案31.根据实施方案1-30中任一项所述的双特异性抗体，其中所述双特异性抗体结合人PD-L1的K D≤1E-09M。
实施方案32.一种分离的核酸，其编码根据实施方案1-31中任一项所述的双特异性抗体。
实施方案33.一种载体，其包含根据实施方案32所述的核酸。
实施方案34.一种宿主细胞，其包含根据实施方案32所述的核酸，或根据实施方案33所述的载体。
实施方案35.一种制备实施方案1-31中任一项所述的双特异性抗体的方法，其包括培养实施方案34所述的宿主细胞，使其表达所述的双特异性抗体，并且在体系中分离纯化所述双特异性抗体。
实施方案36.一种药物组合物，其包含实施方案1-31中任一项所述的双特异性抗体和药学可接受载体。
实施方案37.一种在受试者中治疗癌症的方法，其包括对所述受试者施用治疗有效量的实施方案1-31中任一项所述的双特异性抗体或实施方案36所述的药物组合物。
实施方案38.根据实施方案37所述的在受试者中治疗癌症的方法，其中所述癌症为白血病、淋巴瘤、骨髓瘤、卵巢癌、乳腺癌、子宫内膜癌、结直肠癌、膀胱癌、尿路上皮癌、肺癌、支气管癌、骨癌、***癌、胰腺癌、胃癌、肝癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、***、以及子宫癌、成胶质细胞瘤、成神经管细胞瘤、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、骨髓增生异常综合征、神经胶质瘤、唾液腺癌、胸腺癌、上皮癌、胃食管交接部癌、食管癌、胸膜间皮瘤、鼻咽癌或肉瘤。
具体实施方式
实施例1：抗CD47单克隆抗体的产生
使用CD47蛋白对小鼠的免疫：
为了产生针对CD47的抗体，使用等体积的弗氏完全佐剂(初次免疫))或弗氏不完全佐剂(加强免疫)乳化的重组体hCD47-His Tag蛋白(ACRObiosystems，目录号：CD7-H5227)(50μg/小鼠Balb/c)或hCD47-mFc(ACRObiosystems，目录号：CD7-H52A5)每2周皮下免疫Balb/c小鼠，免疫三次持续6周。第四次免疫采用hCD47-mFc(ACRObiosystems，目录号：CD7-H52A5)蛋白，20μg/只小鼠，肌肉注射(水相佐剂)。细胞融合前三天，通过静脉内注射不含佐剂的抗原来加强小鼠免疫。使用聚乙二醇1500(Polyethylene Glycol 1500；Roche公司，目录号：10783641001)将来自免疫小鼠的脾细胞(1×10 8)与SP2/0骨髓瘤细胞(1.5×10 7)融合。融合后，将细胞以0.1mL/孔分配到96孔板中，并且在37℃、5％CO 2的培养箱中进行孵育。在第1天，通过向每孔添加另外的0.1mL含有血清和补充有2×甲氨蝶呤的HAT培养基来饲养细胞。在第3天和第7天，用0.1mL新鲜的HAT培养基替换来自每孔的0.1mL培养基。通常在第9-14天之间进行筛选，并且通过ELISA针对与hCD47-His Tag(ACRObiosystems公司，目录号：CD7-H5227)反应的抗体测试培养物上清液，筛选阳性孔。
杂交瘤细胞克隆化采用有限稀释法。将挑选出的阳性混合克隆进行有限稀释，一株阳性混合克隆，铺两块96孔板(100cells/板、200cells/板)，将杂交瘤细胞培养在含有10％胎牛血清的RPMI 1640(Hyclone公司，目录号：SH30809.01)中。10天后，待有限稀释后的亚克隆同样经过ELISA试验检测培养物上清，挑取阳性亚克隆。克隆化的杂交瘤细胞保种并用于cDNA的提取。
抗人CD47抗体的cDNA提取和可变区序列获取：
使用RNA提取试剂盒((Takara，目录号：9767)从筛选出的产生hCD47抗体的杂交瘤细胞系中分离总RNA，以总RNA作为模板，根据制造商的说明书，用逆转录试剂盒(Thermo Fisher，目录号：K1652) 合成第一链cDNA。然后利用合成的cDNA，使用简并小鼠IgG引物，通过PCR反应对抗体相关序列进行体外扩增。
将PCR扩增产物在1％琼脂糖/Tris-醋酸盐凝胶中进行电泳分离。从凝胶上切下具有预期大小(重链和轻链大约450bp)的DNA片段并且对其纯化。将3μL纯化的PCR产物克隆到pMD-19T载体(Takara，目录号：6013)中，并且转化到DH5α化学感受态大肠杆菌(Takara，目录号：9057)中。每个连接反应中，随机选择10个阳性克隆，使用M13正向引物进行DNA测序。
从对应的杂交瘤克隆(2B2、2H8、3F10、16E5、14A9)扩增、测序得到五个鼠源单克隆抗体(2B2、2H8、3F10、16E5、14A9抗体)的重链可变区(VH)序列和轻链可变区(VL)序列，序列信息如下：
Figure PCTCN2022122531-appb-000010
嵌合抗体的构建和表达：
通过将小鼠抗体可变区的基因进行化学合成，将每种小鼠抗体的VL区分别连接到人κ链恒定区构建嵌合轻链，将小鼠VH区连接到人IgG4(EU编号S228P)恒定区构建嵌合重链。通过常规基因工程手段分别构建嵌合轻链的表达质粒和嵌合重链的表达质粒，将CHO细胞(200mL体系，在10 6个细胞/mL下)用100μg的每种嵌合重链表达质粒和嵌合轻链表达质粒进行转染并且将其培养6天。然后用蛋白A柱纯化上清液中的嵌合抗体。
构建并表达得到五种嵌合抗体，其嵌合重链和嵌合轻链的全长序列及其编码核酸如下：Xi2H8(嵌合重链及其编码核酸：SEQ ID NOs：89-90；嵌合轻链及其编码核酸：SEQ ID NOs：91-92)、Xi2B2(嵌合重链及其编码核酸：SEQ ID NOs：97-98；嵌合轻链及其编码核酸：SEQ ID NOs：99-100)、Xi3F10(嵌合重链及其编码核酸：SEQ ID NOs：101-102；嵌合轻链及其编码核酸：SEQ ID NOs：103-104)、Xi16E5(嵌合重链及其编码核酸：SEQ ID NOs：93-94；嵌合轻链及其编码核酸：SEQ ID NOs：95-96)、Xi14A9(嵌合重链及其编码核酸：SEQ ID NOs：105-106；嵌合轻链及其编码核酸：SEQ ID NOs：107-108)。
抗体人源化设计：
3F10、14A9和16E5的人鼠嵌合抗体使用CDR移植方法进行人源化(参见，例如，美国专利号5,225,539)。
人源化3F10、14A9和16E5抗体的序列及其编码核酸如下：hz3F10-1.1(重链及其编码核酸：SEQ ID NOs.109-110；轻链及其编码核酸：SEQ ID NOs.111-112)、hz3F10-2.1(重链及其编码核酸：SEQ ID NOs.113-114；轻链及其编码核酸：SEQ ID NOs.115-116)、hz3F10-3.1(重链及其编码核酸：SEQ ID NOs.117-118；轻链及其编码核酸：SEQ ID NOs.119-120)、hz3F10-4.1(重链及其编码核酸：SEQ ID NOs.121-122；轻链及其编码核酸：SEQ ID NOs.123-124)、hz3F10-5.1(重链及其编码核酸：SEQ ID NOs.125-126；轻链及其编码核酸：SEQ ID NOs.127-128)、hz3F10-6.1(重链及其编码核酸：SEQ ID NOs.129-130；轻链及其编码核酸：SEQ ID NOs.131-132)、hz3F10-1.2(重链及其编码核酸：SEQ ID NOs.133-134；轻链及其编码核酸：SEQ ID NOs.135-136)、hz3F10-2.2(重链及其编码核酸：SEQ ID NOs.137-138；轻链及其编码核酸：SEQ ID NOs.139-140)、hz3F10-3.2(重链及其编码核酸：SEQ ID NOs.141-142；轻链及其编码核酸：SEQ ID NOs.143-144)、hz3F10-4.2(重链及其编码核酸：SEQ ID NOs.145-146；轻链及其编码核酸：SEQ ID NOs.147-148)、hz3F10-5.2(重链及其编码核酸：SEQ ID NOs.149-150；轻链及其编码核酸：SEQ ID NOs.151-152)、hz3F10-6.2(重链及其编码核酸：SEQ ID NOs.153-154；轻链及其编码核酸：SEQ ID NOs.155-156)、hz16E5-1.1(重链及其编码核酸：SEQ ID NOs.157-158；轻链及其编码核酸：SEQ ID NOs.159-160)、hz16E5-1.3(重链及其编码核酸：SEQ ID NOs.161-162；轻链及其编码核酸：SEQ ID NOs.163-164)、hz16E5-1.2(重链及其编码核酸：SEQ ID NOs.165-166；轻链及其编码核酸：SEQ ID NOs.167-168)、hz16E5-3.2(重链及其编码核酸：SEQ ID NOs.169-170；轻链及其编码核酸：SEQ ID NOs.171-172)、hz16E5-3.1(重链及其编码核酸：SEQ ID NOs.173- 174；轻链及其编码核酸：SEQ ID NOs.175-176)、hz16E5-3.3(重链及其编码核酸：SEQ ID NOs.177-178；轻链及其编码核酸：SEQ ID NOs.179-180)、hz14A9-2.3(重链及其编码核酸：SEQ ID NOs.181-182；轻链及其编码核酸：SEQ ID NOs.183-184)、hz14A9-2.4(重链及其编码核酸：SEQ ID NOs.185-186；轻链及其编码核酸：SEQ ID NOs.187-188)。
人源化3F10、14A9和16E5抗体的构建和表达：
合成编码人源化3F10、14A9和16E5抗体全长轻链和全长重链的DNA并且将所述DNA克隆到表达载体(包括例如CN107001463A披露的pcDNA3.1载体、CN109422811A披露的pCHO1.0载体等)。将CHO细胞(200mL体系，在10 6个细胞/mL下)用100μg的每种人源化抗体重链表达质粒和轻链表达质粒进行转染并且将其用ExpiCHO培养基(Gibco；货号：A29100-01)在37℃、5％CO 2的培养箱中培养6天。离心分离得到上清液后，然后用蛋白A柱纯化上清液中的人源化抗体。
实施例2：抗CD47抗体的基于Biacore的亲和力分析
通过Biacore分别测量人CD47蛋白(Human CD47 Protein,His Tag；ACRObiosystems公司，目录号：CD7-H5227)和食蟹猴CD47蛋白(Cynomolgus/Rhesus macaque CD47 Protein,His Tag；ACRObiosystems公司，目录号：CD7-C52H1)与抗CD47抗体(嵌合的或人源化的)之间的结合动力学，获得平衡解离常数K D(单位M)；所述Biacore分析在25℃下进行并且以1Hz的数据收集速率进行记录。
将多克隆兔抗小鼠IgG(GE，BR-1008-38)用10mM pH 5.0的乙酸钠进行稀释，并且使用胺偶联试剂盒(GE，BR10050)固定到CM5生物传感器芯片的参考流动池和实验流动池上达到约15000RΜ。在每个循环的开始，将稀释的测试抗体(1.5μg/mL)注射在实验流动池上，持续1分钟以被捕获。这些数据表明，如通过Biacore测量的，抗CD47抗体(嵌合的和人源化的)结合CD47。
表1：抗CD47嵌合抗体与人CD47和食蟹猴CD47的结合亲和力
Figure PCTCN2022122531-appb-000011
表2：抗CD47人源化抗体与人CD47和食蟹猴CD47的结合亲和力
Figure PCTCN2022122531-appb-000012
Figure PCTCN2022122531-appb-000013
实施例3：抗CD47抗体基于促使巨噬细胞吞噬作用的分析
基于流式细胞术测定抗CD47抗体促使细胞表面含有SIRPα的巨噬细胞吞噬肿瘤细胞的能力。
使用单核细胞分离试剂盒(STEMCELL，目录号：19058)从人的外周血单个核细胞(PBMC，赛笠生物，目录号：190056)中分离出单核细胞，铺入24孔板(Greiner bio-one，目录号：662-160)中，每孔5×10 5个细胞，并在100ng/mL刺激因子M-CSF(R&D Systems公司，目录号：216-MC-010)的环境诱导7天，从而诱导成巨噬细胞。将1×10 6个靶细胞HL60(上海细胞所，目录号：TCHu23)悬浮于5μmol/L CFSE(BD公司，目录号：565082)荧光染料(荧光染料用PBS缓冲液稀释到所需浓度)中，37℃水浴锅避光孵育15min，PBS缓冲液洗涤细胞，制备成CFSE标记的HL60细胞；将CFSE标记的HL60细胞分别与10μg/mL、1μg/mL抗CD47抗体(嵌合的抗体见表3，人源化的抗体见表4)于37℃、5％CO 2培养箱中孵育1-2h。PBS缓冲液洗涤三次孵育后的HL60细胞，加入到巨噬细胞中，37℃、5％CO 2培养箱中作用2-4h，PBS缓冲液洗涤三次后，每孔加入260μL PBS、20μL抗APC-CD14(APC Mouse Anti-Human CD14，BD公司，目录号：555399)和20μL PE-CD11b(PE Mouse Anti-Human CD11b/Mac-1，BD公司，目录号：555388)抗体的混合液，4℃避光孵育30min，PBS缓冲液洗涤三次，胰酶(Gibco，目录号：12604-013)消化下来用流式细胞仪(BD公司，型号规格：C6)检测巨噬细胞的吞噬率(％)。
抗CD47嵌合抗体和抗CD47人源化抗体对巨噬细胞吞噬作用的影响参见表3和表4，其中嵌合抗体Xi3F10、Xi16E5、Xi14A9以及抗CD47人源化抗体具有良好的促吞噬作用，人源化抗体hz14A9-2.3、hz14A9-2.4表现更优异。
表3：抗CD47嵌合抗体基于促使巨噬细胞吞噬作用的分析(吞噬率，％)
抗体名称 10μg/mL 1μg/mL
Xi2B2 4.1 7.3
Xi2H8 3.5 7.1
Xi3F10 59.8 65.7
Xi16E5 28.8 11.5
Xi14A9 44.67 54.17
表4：抗CD47人源化抗体基于促使巨噬细胞吞噬作用的分析(吞噬率，％)
抗体名称 10μg/mL 1μg/mL 抗体名称 10μg/mL 1μg/mL
hz3F10-1.1 43.10 41.25 hz3F10-6.2 44.19 49.32
hz3F10-2.1 55.23 47.24 hz16E5-1.1 4.02 14.79
hz3F10-3.1 58.72 50.60 hz16E5-3.1 24.19 6.37
hz3F10-4.1 58.26 50.26 hz16E5-1.2 17.83 5.1
hz3F10-5.1 58.46 50.45 hz16E5-3.2 2.03 3.02
hz3F10-6.1 60.91 47.57 hz16E5-1.3 4.33 3.03
hz3F10-1.2 38.86 55.52 hz16E5-3.3 4.7 11.62
hz3F10-2.2 34.72 44.19 hz14A9-2.3 74.93 71.48
hz3F10-3.2 37.09 47.04 hz14A9-2.4 81.66 81.65
hz3F10-4.2 31.67 46.24 Hu5F9 72.35 67.5
hz3F10-5.2 29.6 47.06      
实施例4：抗CD47抗体对CD4+T细胞的影响
基于流式细胞术的测定方法，测定抗CD47抗体对CD4+T细胞的影响。
使用T细胞分离试剂盒(Miltenyi Biotech，目录号：130-094-131)从人的外周血单个核细胞(PBMC，赛笠生物，目录号：190056)中分离出CD4+T细胞，铺入24孔板(Greiner bio-one，目录号：662106)，每孔5×10 5个细胞，同时分别加入抗CD47抗体(嵌合的抗体见表5，人源化的抗体见表6)使终浓度分别为10μg/mL和1μg/mL，加入1640完全培养基，37℃、5％CO 2培养箱中培养20h，使用凋亡试剂盒(STEMCELL，目录号：556547)室温染色15min，流式仪(BD，型号规格：C6)检测细胞凋亡率(％)，以早期凋亡和晚期凋亡总和计算凋亡率。结果表明抗CD47抗体(嵌合的抗体见表5和人源化的抗体见表6)对T细胞凋亡没有明显影响。
表5：抗CD47嵌合抗体对CD4+T细胞的影响(凋亡率％)
抗体名称 10μg/mL 1μg/mL
Xi2B2 10.3 12.1
Xi2H8 10.5 12.1
Xi3F10 13.6 12.6
Xi16E5 23.1 0.4
Xi14A9 1.34 1.42
表6：抗CD47人源化抗体对CD4+T细胞的影响(凋亡率％)
抗体名称 10μg/mL 1μg/mL 抗体名称 10μg/mL 1μg/mL
hz3F10-1.1 1.94 1.82 hz3F10-6.2 1.68 2.18
hz3F10-2.1 1.7 1.38 hz16E5-1.1 1.88 1.62
hz3F10-3.1 1.88 2 hz16E5-3.1 2.04 1.8
hz3F10-4.1 1.86 1.94 hz16E5-1.2 1.66 1.08
hz3F10-5.1 1.74 1.94 hz16E5-3.2 1.8 1.52
hz3F10-6.1 2.12 1.34 hz16E5-1.3 1.28 1.38
hz3F10-1.2 1.74 1.7 hz16E5-3.3 1.54 1.54
hz3F10-2.2 1.82 1.94 hz14A9-2.3 1.56 1.59
hz3F10-3.2 1.84 1.66 hz14A9-2.4 1.89 1.38
hz3F10-4.2 1.84 1.78 Hu5F9 1.35 1.58
hz3F10-5.2 1.94 1.78 IgG4 1.72 1.81
实施例5：细胞表面抗原数目定量
检测CCRF-CEM-PDL1-20(表达PDL1的CCRF-CEM细胞)和Raji-PDL1(表达PDL1的Raji细胞)细胞表面的PD-L1及CD47抗原表达丰度，使用流式细胞仪(Thermo，Thermo Attune NxT)作为检测仪器，PE荧光定量检测试剂盒PE Phycoerythrin Fluorescence Quantitation Kit(BD biosciences，货号340495)作为定量标准，含藻红蛋白(R-PE)荧光标记后的PE anti-human PD-L1(BD biosciences，目录号557924)、PE anti-human CD47(Biolegend，目录号323108)蛋白作为检测抗体；实验时按照每个样品1×10 6个细胞，加入检测抗体后于100μL体系，37℃反应40分钟后，清洗3次，上机检测每个样品的平均荧光强度(MFI)，计算各细胞系上目标蛋白的表达丰度。表7列出了所检测的相关细胞系上人PD-L1(hPD-L1)和人CD47(hCD47)抗原数目。
表7：细胞表面PD-L1和CD47抗原数目
Figure PCTCN2022122531-appb-000014
Figure PCTCN2022122531-appb-000015
实施例6：anti-PDL1*CD47双特异性抗体的构建和表达纯化
构建的anti-PDL1*CD47双特异性抗体CRS147-3355b由四条多肽链组成，各多肽链的氨基酸和核苷酸序列见表8。对照抗体BM由三条多肽链组成，序列来源于专利申请WO2020259605A1中的SEQ ID NO：1、8、14。
表8：anti-PDL1*CD47双特异性抗体CRS147-3355b的序列
Figure PCTCN2022122531-appb-000016
Figure PCTCN2022122531-appb-000017
化学合成编码抗体各多肽链的DNA序列，并构建重组质粒。具体过程是将编码组成抗体的各多肽链的DNA序列分别***表达载体(例如CN107001463A披露的pcDNA3.1载体，CN109422811A披露的pCHO1.0载体，等)，获得表达相应多肽链的重组表达载体。将编码每种多肽链的重组表达载体按比例共转染ExpiCHO-S细胞(厂家：上海医药工业研究院，目录号：127200005)，转染表达体积为0.1-1L，细胞密度为6E+06cells/mL。转染后将细胞置于ExpiCHO TM培养基(厂家：Thermo，货号：A2910001)中，于32℃、5％CO 2、130rpm条件下表达培养，第10天收取细胞上清液，使用来自GE的AKTA Pure 25L***和MabSelect SuRe LX填料对目标蛋白进行蛋白A亲和层析。洗脱后，使用NanoDrop Lite(Thermo Fisher Scientific)测量洗脱收获的蛋白浓度，SEC-HPLC检测洗脱的蛋白纯度。
为了确认所表达的抗体与示出于表8中的一级氨基酸序列相匹配，通过SDA-PAGE、分子量检测、质谱等分析抗体分子。
纯化结果表明，CRS147-3355b(图3)和BM双抗均具有良好的均一性，SEC-HPLC纯度均>90％。
实施例7：基于SPR的Anti-PDL1*CD47双特异性抗体的亲和力检测
利用表面等离子共振技术(SPR)，通过Biacore 8K***检测HZ5G11单抗(重链和轻链的氨基酸序列为SEQ ID NO：215/216)、HZ14A9单抗(重链和轻链的氨基酸序列为SEQ ID NO：217/218)、CRS147-3355b和BM双特异性抗体的亲和力性质。通过捕获法分别将HZ5G11单抗、HZ14A9单抗、CRS147-3355b和BM双特异性抗体固定到传感芯片上，进样一定浓度梯度的人PD-L1(厂家：百普赛斯，货号：PD1-H52H3)或人CD47(厂家：百普赛斯，货号：CD7-H5227)蛋白抗原溶液，Biacore 8K Control Software 3.0实时采集结合解离曲线，并通过Biacore Insight Evaluation Software 3.0进行数据分析，得出结合速率常数K a、解离速率常数K d和平衡解离常数K D。
表9：基于SPR测定的抗体对人CD47抗原的K D
抗体样品 抗原蛋白 K a(1/Ms) K d(1/s) K D(M)
HZ14A9 hCD47 2.45E+06 1.43E-02 5.85E-09
BM hCD47 2.01E+06 1.73E-03 8.62E-10
CRS147-3355b hCD47 4.22E+06 1.64E-02 3.88E-09
表10：基于SPR测定的抗体对人PD-L1抗原的K D
抗体样品 抗原蛋白 K a(1/Ms) K d(1/s) K D(M)
HZ5G11 hPD-L1 1.51E+06 4.27E-04 2.82E-10
BM hPD-L1 2.47E+06 8.41E-03 3.40E-09
CRS147-3355b hPD-L1 1.69E+06 8.36E-04 4.93E-10
实验结果表明，本发明构建的示例性双特异性抗体CRS147-3355b能有效结合人CD47抗原和人PD-L1抗原。对于人CD47蛋白，BM表现出更强的亲和力，为CRS147-3355b亲和力的4.5倍(表9)，就CD47靶点的分布而言，高亲和力的抗CD47抗体更可能造成潜在的血液安全性问题；如表10所示，对于人PD-L1蛋白，尽管BM的抗PD-L1臂是纳米抗体VHH双价串联，但是其亲和力明显弱于CRS147-3355b(约下降了6.8倍)。相对于BM，CRS147-3355b的分子设计在亲和力的选择、分布以及安全性方面更为合理。
实施例8：Anti-PDL1*CD47双特异性抗体的热稳定性检测
通过Nano DSF(Nano Temper)表征HZ5G11单抗、HZ14A9单抗、CRS147-3355b和BM双特异性抗体的热稳定性质。将待测抗体样品的浓度调整至1.0mg/mL左右，然后用石英毛细管分别吸取少量样品，置于Nano DSF样品悬架之中，以恒定速率1.0℃/min在20.0℃-95.0℃区间程序升温，收集蛋白质样品的光学信号变化，分析蛋白热稳定性。
表11：Anti-PDL1*CD47双特异性抗体的熔解温度
抗体样品 熔解温度Tm1(℃)
CRS147-3355b 68.7
BM 65.7
Nano DSF实验结果显示CRS147-3355b和BM都具有良好的热稳定性，熔解温度Tm1均高于65℃(表11)。其中CRS147-3355b的Tm1值更高，为68.7℃，表明CRS147-3355b具有更好的热稳定性。
实施例9：Anti-PDL1*CD47双特异性抗体与细胞的结合分析
分别取悬浮细胞Raji-PDL1和CCRF-CEM-PDL1-20，1500rpm离心3min收集细胞。加入PBS重悬细胞，细胞计数，向96孔板中加入1.5E+05个细胞/孔，1500rpm离心3min。弃上清，加入8个浓度梯度(起始浓度为400nM，3倍浓度梯度稀释)的抗体样品，每孔100μL，重悬细胞，4℃孵育1h；孵育结束后，1500rpm离心3min，弃上清，每孔加入200μL PBS重悬细胞，重复清洗操作2次。使用PBS稀释二抗RPE-Goat anti human IgG(Jackson ImmunoReseach)，稀释比例为1:200，将稀释后的二抗以100μL/孔加入96孔板，重悬细胞，4℃避光孵育30min；孵育结束后，每孔加入200μL PBS重悬细胞，重复清洗操作2次，再加50μL PBS重悬细胞；流式细胞仪上机检测，设门采集信号。
如图4所示，在CCRF-CEM-PDL1-20细胞中，CRS147-3355b和BM表现出几乎一致的细胞结合行为。而在Raji-PDL1细胞(图5)，CRS147-3355b与BM表现出完全不同的细胞结合行为；相对于BM，CRS147-3355b具有更佳的对肿瘤细胞的亲和力。
实施例10：混合淋巴细胞反应检测Anti-PDL1*CD47双特异性抗体对PD-Ll/PD-1途径的阻断作用
采用混合淋巴细胞反应(MLR)来测试anti-PDL1*CD47双特异性抗体在阻断淋巴细胞效应细胞中的PD-Ll/PD-1途径中的作用。使用CD4+阴性选择分离试剂盒(Mitenyi Biotech，目录号130-091-155)从人PBMC中纯化人CD4+T细胞。使用冻存的PBMC细胞，提取单核细胞，用DC分化试剂刺激5天后，加入成熟试剂孵育2天，使其分化为成熟DC(树突状细胞)。为了建立MLR，对于每个反应，添加1E+05个纯化的CD4+T细胞和1E+04同种异体成熟DC细胞，加入不同浓度的待测抗体，总体积为200μL，检测IFN-γ和IL-2的释放，其中IL-2检测利用试剂盒Human IL-2Flex Set(厂家BD Pharmingen)，IFN-γ检测利用试剂盒hu-IFN-γFlex Set(厂家BD Pharmingen)。
MLR实验中，对照抗体为HZ5G11(anti-PDL1抗体)，在刺激T细胞分泌IL-2(图6A)和IFN-γ(图6B)两种细胞因子作用明显。双特异性抗体样品BM和CRS147-3355b均显示出较强的刺激细胞因子分泌的活性。
实施例11：红细胞凝集实验
通过人红细胞凝集实验表征CRS147-3355b和BM潜在血液安全性质。将红细胞充分清洗，以除去附着在红细胞膜表面的血浆。使用等渗的稀释液将红细胞洗涤3次，前2次2000rpm/min离心5min；末次2000rpm/min离心10min，弃上清。使用PBS将红细胞制成2％细胞悬液，吸取100μL放入96孔U型板中，1500rpm/min离心5min，弃上清。将CRS147-3355b和BM以及同型对照hIgG1样品调整为8个浓度梯度(0，0.1，0.2，0.7，2，6，17，50μg/mL)，96孔U型板加入抗体稀释液，反应总体积为100μL，重悬混匀后于37℃培养箱静置2h，孵育2h后观察结果。如图7所示，双特异性抗体浓度在0-50μg/mL范围内，CRS147-3355b几乎不表现出任何的红细胞凝集能力。而在≥2μg/mL浓度时，BM表现出显著的红细胞凝集现象。红细胞凝集实验表明相对于BM，CRS147-3355b在血液毒性方面更为安全和合理。

Claims (17)

  1. 一种双特异性抗体，其包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含：
    重链可变区，其包含：
    HCDR1，其包含SEQ ID NO：5、1、2、3或4所示的氨基酸序列，
    HCDR2，其包含SEQ ID NO：10、6、7、8或9所示的氨基酸序列，和
    HCDR3，其包含SEQ ID NO：15、11、12、13或14所示的氨基酸序列；和
    轻链可变区，其包含：
    LCDR1，其包含SEQ ID NO：20、16、17、18或19所示的氨基酸序列，
    LCDR2，其包含SEQ ID NO：25、21、22、23或24所示的氨基酸序列，和
    LCDR3，其包含SEQ ID NO：30、26、27、28或32所示的氨基酸序列。
  2. 根据权利要求1所述的双特异性抗体，其中所述第一抗原结合部分包含：
    (v)重链可变区，其包含：
    包含SEQ ID NO：5所示氨基酸序列的HCDR1，
    包含SEQ ID NO：10所示氨基酸序列的HCDR2，和
    包含SEQ ID NO：15所示氨基酸序列的HCDR3；和
    轻链可变区，其包含：
    包含SEQ ID NO：20所示氨基酸序列的LCDR1，
    包含SEQ ID NO：25所示氨基酸序列的LCDR2，和
    包含SEQ ID NO：30所示氨基酸序列的LCDR3；
    (i)重链可变区，其包含：
    包含SEQ ID NO：1所示氨基酸序列的HCDR1，
    包含SEQ ID NO：6所示氨基酸序列的HCDR2，和
    包含SEQ ID NO：11所示氨基酸序列的HCDR3；和
    轻链可变区，其包含：
    包含SEQ ID NO：16所示氨基酸序列的LCDR1，
    包含SEQ ID NO：21所示氨基酸序列的LCDR2，和
    包含SEQ ID NO：26所示氨基酸序列的LCDR3；
    (ii)重链可变区，其包含：
    包含SEQ ID NO：2所示氨基酸序列的HCDR1，
    包含SEQ ID NO：7所示氨基酸序列的HCDR2，和
    包含SEQ ID NO：12所示氨基酸序列的HCDR3；和
    轻链可变区，其包含：
    包含SEQ ID NO：17所示氨基酸序列的LCDR1，
    包含SEQ ID NO：22所示氨基酸序列的LCDR2，和
    包含SEQ ID NO：27所示氨基酸序列的LCDR3；
    (iii)重链可变区，其包含：
    包含SEQ ID NO：3所示氨基酸序列的HCDR1，
    包含SEQ ID NO：8所示氨基酸序列的HCDR2，和
    包含SEQ ID NO：13所示氨基酸序列的HCDR3；和
    轻链可变区，其包含：
    包含SEQ ID NO：18所示氨基酸序列的LCDR1，
    包含SEQ ID NO：23所示氨基酸序列的LCDR2，和
    包含SEQ ID NO：28所示氨基酸序列的LCDR3；或者
    (iv)重链可变区，其包含：
    包含SEQ ID NO：4所示氨基酸序列的HCDR1，
    包含SEQ ID NO：9所示氨基酸序列的HCDR2，和
    包含SEQ ID NO：14所示氨基酸序列的HCDR3；和
    轻链可变区，其包含：
    包含SEQ ID NO：19所示氨基酸序列的LCDR1，
    包含SEQ ID NO：24所示氨基酸序列的LCDR2，和
    包含SEQ ID NO：32所示氨基酸序列的LCDR3。
  3. 一种双特异性抗体，其包含结合CD47的第一抗原结合部分和结合PD-L1的第二抗原结合部分；其中所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO：87、33、35、37、39、41、83或85所示可变区序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含SEQ ID NO：88、34、36、38、40、42、84或86所示可变区序列的LCDR1、LCDR2和LCDR3。
  4. 根据权利要求1-3中任一项所述的双特异性抗体，其中所述第一抗原结合部分的重链可变区包含与SEQ ID NO：33、35、37、39、41、83、85或87所示序列具有至少80％同一性的氨基酸序列；
    或者，所述第一抗原结合部分的轻链可变区包含与SEQ ID NO：34、36、38、40、42、84、86或88所示序列具有至少80％同一性的氨基酸序列；
    或者，所述第一抗原结合部分的重链可变区包含SEQ ID NO：33、35、37、39、41、83、85或87所示的氨基酸序列，所述第一抗原结合部分的轻链可变区包含SEQ ID NO：34、36、38、40、42、84、86或88所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的双特异性抗体，其中所述第一抗原结合部分的重链可变区和轻链可变区选自以下任一项：
    (a)所述的重链可变区包含与SEQ ID NO：33所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：34所示序列具有至少80％同一性的氨基酸序列；
    (b)所述的重链可变区包含与SEQ ID NO：35所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：36所示序列具有至少80％同一性的氨基酸序列；
    (c)所述的重链可变区包含与SEQ ID NO：37所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：38所示序列具有至少80％同一性的氨基酸序列；
    (d)所述的重链可变区包含与SEQ ID NO：39所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：40所示序列具有至少80％同一性的氨基酸序列；
    (e)所述的重链可变区包含与SEQ ID NO：41所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：42所示序列具有至少80％同一性的氨基酸序列；
    (f)所述的重链可变区包含与SEQ ID NO：83所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：84所示序列具有至少80％同一性的氨基酸序列；
    (g)所述的重链可变区包含与SEQ ID NO：85所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：86所示序列具有至少80％同一性的氨基酸序列；
    (h)所述的重链可变区包含与SEQ ID NO：87所示序列具有至少80％同一性的氨基酸序列，所述的轻链可变区包含与SEQ ID NO：88所示序列具有至少80％同一性的氨基酸序列；
    (i)所述的重链可变区包含SEQ ID NO：33所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：34所示的氨基酸序列；
    (j)所述的重链可变区包含SEQ ID NO：35所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：36所示的氨基酸序列；
    (k)所述的重链可变区包含SEQ ID NO：37所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：38所示的氨基酸序列；
    (l)所述的重链可变区包含SEQ ID NO：39所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：40所示的氨基酸序列；
    (m)所述的重链可变区包含SEQ ID NO：41所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：42所示的氨基酸序列；
    (n)所述的重链可变区包含SEQ ID NO：83所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：84所示的氨基酸序列；
    (o)所述的重链可变区包含SEQ ID NO：85所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：86所示的氨基酸序列；
    (p)所述的重链可变区包含SEQ ID NO：87所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：88所示的氨基酸序列；
    (q)所述的重链可变区包含SEQ ID NO：81所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：82所示的氨基酸序列；或者
    (r)所述的重链可变区包含SEQ ID NO：79所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：80所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的双特异性抗体，其中所述第二抗原结合部分包含重链可变区和轻链可变区，其中重链可变区包含：包含SEQ ID NO：199所示氨基酸序列的HCDR1、包含SEQ ID NO：200所示氨基酸序列的HCDR2、和包含SEQ ID NO：201所示氨基酸序列的HCDR3，轻链可变区包含：包含SEQ ID NO：202所示氨基酸序列的LCDR1、包含SEQ ID NO：203所示氨基酸序列的LCDR2、和包含SEQ ID NO：204所示氨基酸序列的LCDR3；或者，
    所述第二抗原结合部分的重链可变区包含SEQ ID NO：205所示的重链可变区序列的HCDR1，HCDR2和HCDR3；所述第二抗原结合部分的轻链可变区包含SEQ ID NO：206所示的轻链可变区序列的LCDR1，HCDR2和LCDR3。
  7. 根据权利要求1-6中任一项所述的双特异性抗体，其中所述第二抗原结合部分的重链可变区包含与SEQ ID NO：205所示序列具有至少85％同一性的氨基酸序列，所述第二抗原结合部分的轻链可变区包含与SEQ ID NO：206所示序列具有至少80％同一性的氨基酸序列；
    优选地，所述的重链可变区包含SEQ ID NO：205所示的氨基酸序列，所述的轻链可变区包含SEQ ID NO：206所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的双特异性抗体，其中所述第一抗原结合部分和第二抗原结合部分各自独立的为Fab、ScFv或ScFab；优选地，所述第一抗原结合部分和第二抗原结合部分均为Fab。
  9. 根据权利要求8所述的双特异性抗体，其中所述第一抗原结合部分和第二抗原结合部分均为Fab，所述第一抗原结合部分和第二抗原结合部分的其中之一具有结构域替换，所述结构域替换为
    重链可变区和轻链可变区彼此替换，或/和
    恒定结构区CH1和CL彼此替换；
    优选地，所述第二抗原结合部分中，恒定结构区CH1和CL彼此替换，使得第二抗原结合部分包含由重链可变区和轻链恒定区构成的肽链和由轻链可变区和重链恒定区构成的肽链。
  10. 根据权利要求1-9中任一项所述的双特异性抗体，其中双特异性抗体还包含由两个Fc多肽构成的Fc域；
    可选地，所述第一抗原结合部分在其C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分在其C端融合至所述Fc域的另一个Fc多肽的N端；
    优选地，所述第一抗原结合部分为Fab且所述第一抗原结合部分在其Fab重链的C端融合至所述Fc域的其中一个Fc多肽的N端，所述第二抗原结合部分为Fab且所述第二抗原结合部分在其Fab重链的C端融合至所述Fc域的另一个Fc多肽的N端。
  11. 根据权利要求10所述的双特异性抗体，其中所述Fc域是IgG Fc域或人IgG Fc域，优选为人IgG1或人IgG4的Fc域；可选地，
    所述Fc域包含促进所述Fc域的两个Fc多肽相缔合的氨基酸取代，或/和
    所述Fc域包含降低Fc域对Fc受体的结合亲和力和/或效应子功能的氨基酸取代，或/和
    所述Fc域包含减少或消除Fc域中的一个Fc多肽的CH3区与A蛋白的结合的氨基酸取代。
  12. 根据权利要求1-11中任一项所述的双特异性抗体，其中所述双特异性抗体包含：
    第一重链，其包含所述第一抗原结合部分的重链可变区；
    第一轻链，其包含所述第一抗原结合部分的轻链可变区；
    第二重链，其包含所述第二抗原结合部分的重链可变区；和
    第二轻链，其包含所述第二抗原结合部分的轻链可变区。
  13. 根据权利要求12所述的双特异性抗体，其中所述第一重链包含与SEQ ID NO：207所示序列具有至少85％同一性的氨基酸序列，所述第一轻链包含与SEQ ID NO：208所示序列具有至少85％同一性的氨基酸序列，所述第二重链包含与SEQ ID NO：209所示序列具有至少85％同一性的氨基酸序列，所述第二轻链包含与SEQ ID NO：210所示序列具有至少85％同一性的氨基酸序列；或者
    所述第一重链包含SEQ ID NO：207所示的氨基酸序列，所述第一轻链包含SEQ ID NO：208所示的氨基酸序列，所述第二重链包含SEQ ID NO：209所示的氨基酸序列，所述第二轻链包含SEQ ID NO：210所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的双特异性抗体，其为二价的。
  15. 根据权利要求1-14中任一项所述的双特异性抗体，其中所述双特异性抗体结合人CD47的K D≤1E-08M；可选地，所述双特异性抗体结合人PD-L1的K D≤1E-09M。
  16. 一种药物组合物，其包含权利要求1-15中任一项所述的双特异性抗体和药学可接受载体。
  17. 一种在受试者中治疗癌症的方法，其包括对所述受试者施用治疗有效量的权利要求1-15中任一项所述的双特异性抗体或权利要求16所述的药物组合物；优选地，所述癌症为白血病、淋巴瘤、骨髓瘤、卵巢癌、乳腺癌、子宫内膜癌、结直肠癌、膀胱癌、尿路上皮癌、肺癌、支气管癌、骨癌、***癌、胰腺癌、胃癌、肝癌、胆囊癌、胆管癌、食道癌、肾细胞癌、甲状腺癌、头颈癌、睾丸癌、内分泌腺癌、肾上腺癌、脑下垂体癌、皮肤癌、软组织癌、血管癌、脑癌、神经癌、眼癌、脑膜癌、口咽癌、下咽部癌、***、以及子宫癌、成胶质细胞瘤、成神经管细胞瘤、星形细胞瘤、胶质瘤、脑膜瘤、胃泌素瘤、成神经细胞瘤、黑色素瘤、骨髓增生异常综合征、神经胶质瘤、唾液腺癌、胸腺癌、上皮癌、胃食管交接部癌、食管癌、胸膜间皮瘤、鼻咽癌或肉瘤。
PCT/CN2022/122531 2021-09-30 2022-09-29 针对免疫检查点的双特异性抗体 WO2023051680A1 (zh)

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