WO2021170082A1 - 抗cd47/抗pd-l1抗体及其应用 - Google Patents

抗cd47/抗pd-l1抗体及其应用 Download PDF

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WO2021170082A1
WO2021170082A1 PCT/CN2021/078132 CN2021078132W WO2021170082A1 WO 2021170082 A1 WO2021170082 A1 WO 2021170082A1 CN 2021078132 W CN2021078132 W CN 2021078132W WO 2021170082 A1 WO2021170082 A1 WO 2021170082A1
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seq
amino acid
acid sequence
antibody
variable region
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French (fr)
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肖扬
朱永强
赵立文
吴文明
周果
罗成
李雪
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南京圣和药业股份有限公司
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Priority to EP21760510.4A priority Critical patent/EP4112647A4/en
Priority to US17/802,904 priority patent/US20230090014A1/en
Publication of WO2021170082A1 publication Critical patent/WO2021170082A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the technical field of antibody medicines, in particular to anti-CD47/anti-PD-L1 antibodies, pharmaceutical compositions containing anti-CD47/anti-PD-L1 antibodies and applications thereof.
  • CD47 protein also known as integrin-related protein (IAP), belongs to a five-pass transmembrane glycoprotein in the IgG superfamily and is widely expressed in different tissues and cells.
  • CD47 can bind to the ligand TSP-1 or SIRP ⁇ to regulate different cell functions, including cell migration, adhesion, apoptosis, axon extension, cytokine production and T cell activation.
  • SIRP ⁇ is a transmembrane protein containing a typical immune receptor tyrosine inhibitory motif (ITIM), which is mainly expressed on the surface of myeloid hematopoietic cell membranes, such as macrophages and dendritic cells.
  • ITIM immune receptor tyrosine inhibitory motif
  • CD47 After CD47 binds to SIRP ⁇ , it leads to phosphorylation of ITIMs, which recruits SHP-1/SHP-2, which in turn inhibits the accumulation of myosin IIA in the phagocytic synapse, and ultimately inhibits the phagocytic function of phagocytes.
  • the "immune escape" of tumor cells is considered to be the main mechanism of tumor occurrence, development and drug resistance.
  • tumor cells interact with SIRP ⁇ on the surface of macrophages, which can significantly inhibit the phagocytic activity of macrophages and avoid being engulfed by macrophages.
  • SIRP ⁇ an enzyme that catalyzes the phagocytic activity of macrophages.
  • it can eliminate the immunosuppression or immune tolerance caused by tumors, and effectively kill tumor cells. This provides a very strong theoretical basis for CD47 tumor immune targeted therapy.
  • CD47 blocking antibodies are considered to be the most promising tumor treatment options.
  • the effectiveness of human CD47 blocking monoclonal antibodies has been confirmed in a variety of preclinical models.
  • red blood cells and platelets also express CD47 molecules, when antibodies block the interaction between CD47 and SIRP ⁇ , these cells may lose the protection of the "don't eat me" signal and be swallowed by macrophages. Therefore, avoiding the side effects of anti-CD47 antibodies, such as platelet degradation, red blood cell agglutination, red blood cell depletion, anemia, etc. is also an important point to consider when applying anti-CD47 antibodies.
  • PD-L1 Programmed death ligand 1
  • CD274 cluster of differentiation 274
  • B7-H1 B7 homolog 1
  • PD-L1 is a 40kDa type 1 transmembrane protein that is used in specific events (such as pregnancy).
  • Tissue allogeneic transplantation, autoimmune diseases and other disease states (such as hepatitis) play a major role in the process of suppressing the immune system.
  • the binding of PD-L1 to PD-1 or B7.1 delivers inhibitory signals, which reduce the proliferation of CD8 + T cells in lymph nodes, and the complement of PD-1 can also be further enhanced by the gene Bcl-2
  • the lower regulation-mediated apoptosis to control the accumulation of foreign antigen-specific T cells in lymph nodes.
  • Upregulation of PD-L1 has been shown to allow cancer to evade the host immune system. Analysis of tumor samples from patients with renal cell carcinoma found that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and increased risk of death. Many PD-L1 inhibitors are being developed as immuno-oncology therapies and are showing good results in clinical trials.
  • the CD47-SIRP ⁇ signaling pathway not only activates innate immunity, but macrophages also present tumor antigens to CD8+T and CD4+T cells, and further kill tumors by promoting T cell activation. Therefore, it is possible to develop a bifunctional fusion protein targeting CD47 and PD-L1.
  • the fusion protein contains a CD47 binding part and a PD-L1 binding part, which can both block the binding of PD-L1 and PD-1 and block the binding of PD-L1 and PD-1. Cut off the combination of CD47 and SIRP ⁇ , bridging the innate immune and acquired immune signaling pathways, thereby having better anti-tumor activity, tumor targeting and lower red blood cell toxicity.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which comprises an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or an antigen-binding fragment thereof comprises a first The heavy chain variable region and/or the first light chain variable region, wherein the first heavy chain variable region comprises the complementarity determining region 1 (H1CDR1) of the first heavy chain variable region, the first heavy chain variable region Complementarity determining region 2 (H1CDR2) and/or complementarity determining region 3 (H1CDR3) of the first heavy chain variable region, said first light chain variable region comprising complementarity determining region 1 (H1CDR3) of the first light chain variable region L1CDR1), the complementarity determining region 2 (L1CDR2) of the first light chain variable region and/or the complementarity determining region 3 (L1CDR3) of the first light chain variable region; and the anti-PD-L1 antibody
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or an antigen-binding fragment thereof
  • the antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the following group:
  • the first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the following group:
  • the anti-CD47 antibody or antigen-binding fragment thereof has:
  • the H1CDR1, H1CDR2, and H1CDR3 are respectively SEQ ID NOs: 1, 2 and 3 or the first heavy chain of an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2 and 3.
  • the variable region and the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NOs: 12, 13, and 14, or the amino acid sequence of SEQ ID NO: 12, 13 and 14 having at least 85% sequence identity.
  • the H1CDR1, H1CDR2, and H1CDR3 are respectively SEQ ID NOs: 10, 2 and 11 or the first heavy chain of an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, 2 and 11.
  • the variable region and the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NOs: 12, 13, and 14, or the amino acid sequence of SEQ ID NO: 12, 13 and 14 having at least 85% sequence identity.
  • the H1CDR1, H1CDR2, and H1CDR3 are respectively SEQ ID NOs: 4, 5, and 6, or the first heavy chain of an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, 5, and 6.
  • the variable region and the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 15, 16, and 17, or the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, 16 and 17.
  • the H1CDR1, H1CDR2, and H1CDR3 are respectively SEQ ID NOs: 7, 8 and 9 or the first heavy chain of an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, 8 and 9
  • the variable region and the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 18, 19, and 20 or the amino acid sequence of SEQ ID NO: 18, 19, and 20 that has at least 85% sequence identity.
  • a light chain variable region is respectively SEQ ID NOs: 7, 8 and 9 or the first heavy chain of an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, 8 and 9
  • the variable region and the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 18, 19, and 20 or the amino acid sequence of SEQ ID NO: 18, 19, and 20 that has at least 85% sequence identity.
  • a light chain variable region is respectively SEQ ID NOs: 7, 8 and 9 or the first heavy chain
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • amino acid sequence shown in (b1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (b1);
  • amino acid sequence of the first light chain variable region is selected from:
  • amino acid sequence shown in (b4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (b4);
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 21, SEQ ID NO: 21 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 21 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 21, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 24, and SEQ ID NO: 24 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 24 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 24;
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 22, SEQ ID NO: 22 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 22 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 22, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 25, and SEQ ID NO: 25 is substituted, deleted or added by one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 25 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 25;
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 23, SEQ ID NO: 23 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 23, or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 23, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 26, and SEQ ID NO: 26 is substituted, deleted or added by one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 26 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 26;
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 27, SEQ ID NO: 27 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 27 or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 27, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 24, and SEQ ID NO: 24 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 24 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 24; or
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 23, SEQ ID NO: 23 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 23, or An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 23, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 28, and SEQ ID NO: 28 is substituted, deleted or added by one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 28 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 28.
  • the anti-CD47 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, which comprises a first heavy chain variable region and a first heavy chain variable region.
  • a light chain variable region in which:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • amino acid sequence of the first light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c4);
  • the anti-CD47 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, which comprises a first heavy chain variable region and a first heavy chain variable region.
  • a light chain variable region where
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 29, SEQ ID NO: 29 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 29.
  • An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 29, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 32, and SEQ ID NO: 32 is substituted, deleted or added by one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 32 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 32;
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, SEQ ID NO: 30 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 30.
  • An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 33, and SEQ ID NO: 33 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 33 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 33; or
  • the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 31, SEQ ID NO: 31 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 31.
  • An amino acid sequence having at least 85% sequence identity with SEQ ID NO: 31 and the amino acid sequence of the first light chain variable region is SEQ ID NO: 34, and SEQ ID NO: 34 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 34 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 34.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the first heavy chain variable region includes H1CDR1, H1CDR2, and H1CDR3, and its amino acid sequence is SEQ ID NO: 4, 5, and 6, or has the same amino acid sequence as SEQ ID NO: 4, 5, and 6. Amino acid sequences with at least 85% sequence identity; and
  • the first light chain variable region includes L1CDR1, L1CDR2, and L1CDR3, and its amino acid sequence is SEQ ID NO: 15, 16, and 17, or the amino acid sequence shown in SEQ ID NO: 15, 16 and 17. Amino acid sequence with at least 85% sequence identity.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody Or the antigen-binding fragment thereof is as defined in the above embodiments; and the anti-PD-L1 antibody or the antigen-binding fragment thereof comprises a second heavy chain variable region and/or a second light chain variable region, wherein the second The heavy chain variable region includes the complementarity determining region 1 (H2CDR1) of the second heavy chain variable region, the complementarity determining region 2 (H2CDR2) of the second heavy chain variable region and/or the complementarity determination of the second heavy chain variable region Region 3 (H2CDR3), the second light chain variable region includes complementarity determining region 1 (L2CDR1) of the second light chain variable region, complementarity determining region 2 (L2CDR2) of the second light chain variable region and/or The complementarity
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47/anti-PD-L1 antibody
  • the CD47 antibody or antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • the second heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the following group:
  • (A5) an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (A1), (A2), (A3) or (A4);
  • the second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the following group:
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:
  • the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • the first heavy chain variable region includes H1CDR1, H1CDR2, and H1CDR3, and its amino acid sequence is SEQ ID NO: 4, 5, and 6, or has the same amino acid sequence as SEQ ID NO: 4, 5, and 6. Amino acid sequences with at least 85% sequence identity;
  • the first light chain variable region includes L1CDR1, L1CDR2, and L1CDR3, and its amino acid sequence is SEQ ID NO: 15, 16, and 17, or the amino acid sequence shown in SEQ ID NO: 15, 16 and 17. Amino acid sequences with at least 85% sequence identity; and
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • the second heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the following group:
  • (A5) an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (A1), (A2), (A3) or (A4);
  • the second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the following group:
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody Or an antigen-binding fragment thereof comprising an amino acid sequence of which the H1CDR1, H1CDR2, and H1CDR3 are SEQ ID NO: 4, 5, and 6, respectively, or the amino acid sequence shown in SEQ ID NO: 4, 5, and 6 having at least 85% sequence identity
  • the first heavy chain variable region of the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 15, 16 and 17, or the amino acid sequence shown in SEQ ID NO: 15, 16 and 17 has at least 85% sequence identity
  • the first light chain variable region of a sexual amino acid sequence; and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2, and H2CDR3 are respectively SEQ ID NO: 75,
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody Or an antigen-binding fragment thereof comprising an amino acid sequence of which the H1CDR1, H1CDR2, and H1CDR3 are SEQ ID NO: 4, 5, and 6, respectively, or the amino acid sequence shown in SEQ ID NO: 4, 5, and 6 having at least 85% sequence identity
  • the first heavy chain variable region of the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 15, 16 and 17, or the amino acid sequence shown in SEQ ID NO: 15, 16 and 17 has at least 85% sequence identity
  • the first light chain variable region of a sexual amino acid sequence; and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 81,
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody Or an antigen-binding fragment thereof comprising an amino acid sequence of which the H1CDR1, H1CDR2, and H1CDR3 are SEQ ID NO: 4, 5, and 6, respectively, or the amino acid sequence shown in SEQ ID NO: 4, 5, and 6 having at least 85% sequence identity
  • the first heavy chain variable region of the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 15, 16 and 17, or the amino acid sequence shown in SEQ ID NO: 15, 16 and 17 has at least 85% sequence identity
  • the first light chain variable region of a sexual amino acid sequence; and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 87,
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, which includes an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody Or an antigen-binding fragment thereof comprising an amino acid sequence of which the H1CDR1, H1CDR2, and H1CDR3 are SEQ ID NO: 4, 5, and 6, respectively, or the amino acid sequence shown in SEQ ID NO: 4, 5, and 6 having at least 85% sequence identity
  • the first heavy chain variable region of the L1CDR1, L1CDR2, and L1CDR3 are respectively SEQ ID NO: 15, 16 and 17, or the amino acid sequence shown in SEQ ID NO: 15, 16 and 17 has at least 85% sequence identity
  • the first light chain variable region of a sexual amino acid sequence; and the anti-PD-L1 antibody or antigen-binding fragment thereof comprising the H2CDR1, H2CDR2 and H2CDR3 are respectively SEQ ID NO: 93,
  • the anti-CD47 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are each independently a murine antibody, Chimeric antibodies, humanized antibodies, or fully human antibodies.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, in:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • amino acid sequence shown in (b1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (b1);
  • amino acid sequence of the first light chain variable region is selected from:
  • amino acid sequence shown in (b4) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (b4);
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • amino acid sequence of the second heavy chain variable region is selected from:
  • (B2) The amino acid sequence shown in (B1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (B1);
  • amino acid sequence of the second light chain variable region is selected from:
  • the amino acid sequence shown in (B4) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (B4);
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 22, and SEQ ID NO: 22 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 22 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 22, and the first light chain variable region The amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 25 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 25 or having at least 85 amino acid sequence with SEQ ID NO: 25 Amino acid sequence of% sequence identity.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 22, and SEQ ID NO: 22 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 22 or having at least 85% sequence identity with SEQ ID NO: 22 and the H1CDR1, H1CDR2 and H1CDR3 are as SEQ ID NO: 4 , 5, and 6, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 25, SEQ ID NO: 25 is obtained by substituting, deleting or adding one or more amino acids and and
  • the amino acid sequence of SEQ ID NO: 25 is functionally identical or has at least 85% sequence identity with SEQ ID NO: 25 and the L1 CDR1, L1 CDR2, and L1 CDR3 are the amino acid sequences shown in SEQ ID NOs: 15, 16 and 17.
  • the anti-CD47/anti-PD-L1 antibody according to the present invention wherein the anti-CD47 antibody or anti-PD-L1 antibody may be a murine antibody, which also contains murine IgG1, IgG2, and IgG3. Or the heavy chain constant region of IgG4 or a variant thereof, and the light chain constant region of a murine kappa chain or a variant thereof.
  • the anti-CD47/anti-PD-L1 antibody according to the present invention wherein the anti-CD47 murine antibody further contains the heavy chain constant region of murine IgG1 or IgG2 or a variant thereof, and murine origin The light chain constant region of a kappa chain or a variant thereof.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:
  • amino acid sequence of the first heavy chain variable region is selected from:
  • amino acid sequence of the first light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (c4);
  • the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:
  • amino acid sequence of the second heavy chain variable region is selected from:
  • (C2) The amino acid sequence shown in (C1) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (C1);
  • (C3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (C1);
  • amino acid sequence of the second light chain variable region is selected from:
  • the amino acid sequence shown in (C4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (C4);
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acid of the first light chain variable region
  • the sequence is SEQ ID NO: 33
  • SEQ ID NO: 33 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 33 or having at least 85% sequence with SEQ ID NO: 33 Identical amino acid sequence
  • the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 110, 111, 112, 113 or 114, SEQ ID NO: 110, 111, 112, 113 Or 114 is the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acid of the first light chain variable region
  • the sequence is SEQ ID NO: 33
  • SEQ ID NO: 33 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 33 or having at least 85% sequence with SEQ ID NO: 33 Identical amino acid sequence
  • the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 119, 120, 121, 122 or 123, SEQ ID NO: 119, 120, 121, 122 Or 123 is the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids and having the same function as
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted, deleted or The amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85% sequence identity with SEQ ID NO: 30 and the H1CDR1, H1CDR2 and H1CDR3 are as SEQ ID NO: 4, 5.
  • amino acid sequence shown in 6, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 33
  • SEQ ID NO: 33 is obtained by substituting, deleting or adding one or more amino acids and is the same as SEQ ID NO:33 functionally identical amino acid sequence or SEQ ID NO:33 has at least 85% sequence identity and said L1CDR1, L1CDR2 and L1CDR3 are as shown in SEQ ID NO:15, 16 and 17
  • the amino acid sequence of the double heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 110, 111, 112, 113 or 114, and SEQ ID NO: 110, 111, 112, 113 or 114 is substituted, deleted or added one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 110, 111, 112, 113 or 114 or having at least 85% sequence identity with SEQ ID NO: 110, 111, 112, 113 or 114 and said The amino acid sequences of H2CDR1, H2CDR2 and H
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted, deleted or The amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85% sequence identity with SEQ ID NO: 30 and the H1CDR1, H1CDR2 and H1CDR3 are as SEQ ID NO: 4, 5.
  • amino acid sequence shown in 6, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 33
  • SEQ ID NO: 33 is obtained by substituting, deleting or adding one or more amino acids and is the same as SEQ ID NO:33 functionally identical amino acid sequence or SEQ ID NO:33 has at least 85% sequence identity and said L1CDR1, L1CDR2 and L1CDR3 are as shown in SEQ ID NO:15, 16 and 17
  • the amino acid sequence of the double heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 110, 111, 112, 113 or 114, and SEQ ID NO: 110, 111, 112, 113 or 114 is substituted, deleted or added one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 110, 111, 112, 113 or 114 or having at least 85% sequence identity with SEQ ID NO: 110, 111, 112, 113 or 114 and said The amino acid sequences of H2CDR1, H2CDR2 and H
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted, deleted or The amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO: 30 and
  • the H1CDR1, H1CDR2, and H1CDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 33, and SEQ ID NO: 33 is substituted ,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 33 or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence with SEQ ID NO: 33 Identity and the amino acid sequence of the L1CDR1, L1C
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO: 30
  • the H1CDR1, H1CDR2, and H1CDR3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, and the amino acid sequence of the first light chain variable region is SEQ ID NO: 33, SEQ ID NO: 33
  • the present invention provides an anti-CD47/anti-PD-L1 humanized antibody, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, so
  • the light chain includes a human-derived kappa, lambda chain or a light chain constant region of a variant thereof.
  • the murine anti-CD47/anti-PD-L1 antibody may further comprise the light chain constant region of murine ⁇ , ⁇ chain or a variant thereof, and/or further comprise murine origin The heavy chain constant region of IgG1, IgG2, IgG3 or IgG4 or variants thereof.
  • the light chain of the anti-CD47 antibody or antigen-binding fragment thereof further comprises a murine ⁇ , ⁇ chain or a mutant sequence thereof.
  • Light chain constant region The antibody heavy chain of the anti-CD47 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or a mutant sequence thereof, and preferably comprises a human IgG1, IgG2, IgG4 heavy chain constant region.
  • the anti-CD47 humanized antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2, IgG3 or IgG4 or variants thereof.
  • the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG1, IgG2, IgG4 or variants thereof, and human kappa chain or variants thereof The constant region of the light chain.
  • the antibody heavy chain of the anti-PD-L1 antibody or antigen-binding fragment thereof further comprises murine IgG1, IgG2, IgG3, IgG4 Or the heavy chain constant region of its mutant sequence, preferably comprising the heavy chain constant region of human IgG or its mutant sequence;
  • the antibody light chain of the anti-PD-L1 antibody or its antigen-binding fragment further comprises murine ⁇ , ⁇ chain or The mutated sequence of the light chain constant region.
  • the anti-PD-L1 humanized antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • the anti-CD47 humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG4 or its variants, and the light chain constant of human ⁇ chain or its variants. Area.
  • the present invention provides an anti-CD47/anti-PD-L1 antibody, wherein the anti-CD47 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are respectively Fab, Fv, sFv or F (ab) 2 .
  • the anti-CD47/anti-PD-L1 antibody provided by the present invention is scF(ab) 2 .
  • the anti-CD47/anti-PD-L1 antibody in the above embodiment of the present invention is an anti-CD47/anti-PD-L1 bispecific antibody.
  • the bispecific antibody is a human antibody or a humanized antibody.
  • one of the binding specificities is for CD47 and the other binding specificity is for any other antigen.
  • one of the binding specificities is for CD47 and the other binding specificity is for PD-L1.
  • the bispecific antibody can bind to two different epitopes of CD47.
  • the bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD47.
  • bispecific antibodies of the present invention possess a CD47 binding arm and a cytotoxic agent binding arm, such as saponin, anti-interferon- ⁇ , vinca alkaloids, ricin A chain, methotrexate, or radioisotope hapten .
  • the bispecific antibodies of the present invention can be prepared as full-length antibodies or antibody fragments (for example, F(ab') 2 bispecific antibodies).
  • bispecific antibodies Methods of preparing bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random allocation of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, of which only one molecule has the correct bispecific structure. The purification of the correct molecule is usually carried out through an affinity chromatography step, which is quite troublesome and the product yield is low. Similar methods are disclosed in WO93/08829 and Traunecker et al., EMBOJ. 10:3655 (1991).
  • antibody variable regions with the desired binding specificity are fused with immunoglobulin constant region sequences.
  • the fusion is performed with an immunoglobulin heavy chain constant region comprising at least part of the hinge, CH2, and CH3 regions.
  • the first heavy chain constant region (CH1) containing the site necessary for binding to the light chain is present in at least a portion of the fusion.
  • the DNA encoding the immunoglobulin heavy chain fusion fragment and the immunoglobulin light chain (if necessary) are inserted into different expression vectors and co-transfected into a suitable host organism.
  • the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm. (Providing a second binding specificity) composition. Since the immunoglobulin light chain is only present in half of the bispecific molecule to provide a convenient way of separation, the asymmetric structure was found to facilitate the separation of the desired bispecific substance from the undesired immunoglobulin chain composition . This method is disclosed in WO 94/04690. For further information on the production of bispecific antibodies, see, for example, Sureshetal., Methods in Enzymology 121:210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture.
  • the interface contains at least a portion of the CH3 domain of the antibody constant region.
  • one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (such as tyrosine or tryptophan).
  • a compensating "cavity" of the same or similar size as the large side chain is created on the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other undesirable end products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one heterologous conjugated antibody can be conjugated to avidin, and another heterologous conjugated antibody can be conjugated to biotin.
  • Any convenient cross-linking method can be used to prepare heteroconjugate antibodies. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with many crosslinking techniques.
  • the bispecific antibodies of the present invention can be produced from antibody fragments.
  • chemical ligation techniques can be used to prepare bispecific antibodies.
  • Brennanetal., Science 229:81 (1985) describes a method for proteolytic cleavage of intact antibodies to generate F(ab') 2 fragments. These fragments are decomposed in the presence of the dithiol complexing agent sodium arsenite (to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds).
  • the resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives is restored to Fab'-thiol through the reduction of mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody.
  • TAB thionitrobenzoate
  • Fab'-SH fragments can be recovered directly from E. coli, and these fragments can be chemically coupled to form bispecific antibodies.
  • Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule.
  • Each Fab' fragment is separately secreted by E. coli and undergoes directed chemical coupling in vitro to form bispecific antibodies.
  • the bispecific antibody fragments of the invention can be produced and isolated directly from recombinant cell culture.
  • a leucine zipper can be used to generate bispecific antibodies (Kostelnyetal., J. Immunol. 148(5):1547-1553 (1992)).
  • the leucine zipper peptides from Fos and Jun proteins were linked to the Fab' parts of two different antibodies by gene fusion.
  • the antibody homodimer is broken down in the hinge region to form monomers, and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers.
  • the diabody technology provides other mechanisms for preparing bispecific antibody fragments.
  • the bispecific antibody fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker that is too short to allow pairing between two domains on the same chain . Therefore, the VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on the other fragment, thereby forming two antigen binding sites.
  • bispecific antibody fragments can be constructed by using single chain Fv (sFv) dimers.
  • the present invention encompasses multivalent antibodies with more than two valences, for example, trispecific antibodies can be prepared.
  • Multivalent antibodies can be internalized (and/or alienated) by cells expressing the antigen to which the antibody binds faster than bivalent antibodies.
  • the antibody of the present invention may be a multivalent antibody having three or more antigen binding sites (for example, a tetravalent antibody) that can be easily produced by recombinant expression of a nucleic acid encoding an antibody polypeptide chain.
  • Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites.
  • the dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • the antibody will contain the Fc region and three or more antigen binding sites at the amino terminus of the Fc region.
  • the multivalent antibody contains (or consists of) three to about eight antigen binding sites.
  • the multivalent antibody contains four antigen binding sites.
  • the multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein the polypeptide chain comprises two or more variable regions.
  • the multivalent antibody of the present invention may further comprise at least two (for example, four) light chain variable region polypeptides.
  • the multivalent antibody of the present invention may comprise, for example, about two to about eight light chain variable region polypeptides.
  • the light chain variable region polypeptide of the present invention includes a light chain variable region, and optionally further includes a CL domain.
  • one of the CD47 targeting moiety and the PD-L1 targeting moiety may be a full-length antibody, and the other may be a heavy chain CDR, light An antigen-binding fragment of a chain CDR or a combination thereof (e.g., scFv).
  • the full-length antibody targeting one of the CD47 and PD-L1 proteins and the antigen-binding fragment targeting the other protein can be chemically linked (for example, covalently linked) directly or through a peptide linker.
  • the antigen-binding fragment (e.g., scFv) can be directly or through a peptide linker to the N-terminus of the full-length antibody (e.g., the N-terminus of the light or heavy chain of the full-length antibody), the C-terminus of the full-length antibody (e.g., the full-length antibody).
  • the C-terminus of the heavy chain (or Fc or CH3 domain) or both are connected.
  • the bispecific antibody may comprise a full-length anti-CD47 antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker between them.
  • the bispecific antibody may comprise a full-length anti-CD47 antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker between them.
  • the scFv contained in the bispecific antibody may contain a heavy chain variable region and a light chain variable region in any order.
  • the scFv contained in the bispecific antibody may include a heavy chain variable region and a light chain variable region in the direction from N-terminus to C-terminus and optionally a peptide linker between them, or alternatively Specifically, the scFv contained in the bispecific antibody may include a light chain variable region and a heavy chain variable region in the direction from the N-terminus to the C-terminus, and optionally a peptide linker between them.
  • the peptide linker may include, for example, Gly, Asn, and/or Ser residues, and may also include neutral amino acids, such as Thr and/or Ala.
  • the amino acid sequence suitable for the peptide linker may be those known in the related art.
  • the length of the peptide linker can be determined differently within such a limit that the function of the fusion protein is not affected.
  • the peptide linker can be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 selected from one or more of the group consisting of Gly, Asn, Ser, Thr, and Ala .
  • the peptide linker can be represented as (GmSl)n (m, l, and n are independently an integer from about 1 to about 10, particularly an integer from about 2 to about 5).
  • the PD-L1 targeting moiety and the CD47 targeting moiety may both be full-length antibodies or antigen-binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof.
  • the bispecific antibody may be in the form of a heterodimer, comprising a first arm and a second arm, the first arm including a pair of first heavy chains targeting one of CD47 and PD-L1 And the first light chain, the second arm includes a pair of a second heavy chain and a second light chain that target the other.
  • the full-length antibody may be in the form of a full-length immunoglobulin (e.g., IgG, IgM, IgA, IgE, or IgD, such as human IgG, human IgM, human IgA, human IgE, or human IgD), and an antigen-binding fragment It can be selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, scFv, scFab, single-chain antibody, sdFv and the like.
  • Fab fragment antigen-binding fragment
  • the full-length antibody may be in the form of a full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment may be a scFv.
  • the antibodies described herein may contain flexible linker sequences, or may be modified to add functional moieties (e.g., PEG, drugs, toxins, or labels).
  • functional moieties e.g., PEG, drugs, toxins, or labels.
  • the structure of the anti-CD47 antibody or antigen-binding fragment thereof is (VL-CL)-peptide linker-(VH)- IgG4CH
  • the structure of the anti-PD-L1 antibody or antigen-binding fragment thereof is (VL-CL)-peptide linker-(VH)-IgG4CH
  • the peptide linker is in the form of (GGGGS)n, where n is 1-12, preferably 3-10, more preferably 6-8, such as 6, 7, 8 GGGGS repeats.
  • the IgG4CH in the (VL-CL)-peptide linker-(VH)-IgG4CH of the CD47 portion contains S228P, L235E, Y349C, T366S, L368A, Y407V mutations to form a "Hole" structure
  • the IgG4CH segment of the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 part is an IgG4CH segment containing S228P, L235E, T366W, and S354C mutations to form a "Knob" structure.
  • the amino acid sequence of VL in the CD47-targeted (VL-CL)-peptide linker-(VH)-IgG4CH is SEQ ID NO: 33, and the amino acid sequence of CL is SEQ ID NO: 131 ,
  • the amino acid sequence of VH is SEQ ID NO: 30, the amino acid sequence of IgG4CH is SEQ ID NO: 133; and/or the VL in the (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 part
  • the amino acid sequence of is SEQ ID NO: 116
  • the amino acid sequence of CL is SEQ ID NO: 131
  • the amino acid sequence of VH is SEQ ID NO: 112
  • the amino acid sequence of IgG4CH is SEQ ID NO: 132.
  • the amino acid sequence of VL in the CD47-targeted (VL-CL)-peptide linker-(VH)-IgG4CH is SEQ ID NO: 33, and the amino acid sequence of CL is SEQ ID NO: 131, the amino acid sequence of VH is SEQ ID NO: 30, the amino acid sequence of IgG4CH is SEQ ID NO: 133; and/or the (VL-CL)-peptide linker-(VH)-IgG4CH in the PD-L1 portion
  • the amino acid sequence of VL is SEQ ID NO: 126
  • the amino acid sequence of CL is SEQ ID NO: 131
  • the amino acid sequence of VH is SEQ ID NO: 123
  • the amino acid sequence of IgG4CH is SEQ ID NO: 132.
  • Another aspect of the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3 selected from the following group:
  • (A5) an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (A1), (A2), (A3) or (A4);
  • the light chain variable region comprises CDR1, CDR2 and CDR3 selected from the following group:
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises:
  • the CDR1, CDR2 and CDR3 are respectively SEQ ID NOs: 75, 76 and 77 or heavy chain variable regions with amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 75, 76 and 77 , And the CDR1, CDR2, and CDR3 are respectively SEQ ID NO: 78, 79 and 80 or the light chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 78, 79 and 80.
  • Variable area are respectively SEQ ID NOs: 75, 76 and 77 or heavy chain variable regions with amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 75, 76 and 77 .
  • the CDR1, CDR2 and CDR3 are respectively SEQ ID NOs: 87, 88 and 89 or heavy chain variable regions with amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 87, 88 and 89
  • the CDR1, CDR2, and CDR3 are respectively SEQ ID NO: 90, 91 and 92 or the light chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 90, 91 and 92.
  • the CDR1, CDR2 and CDR3 are respectively SEQ ID NOs: 93, 94 and 95 or heavy chain variable regions with amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 93, 94 and 95
  • the CDR1, CDR2, and CDR3 are respectively SEQ ID NO: 96, 97 and 98 or the light chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 96, 97 and 98.
  • Variable area are respectively SEQ ID NOs: 93, 94 and 95 or heavy chain variable regions with amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 93, 94 and 95 .
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • (B2) The amino acid sequence shown in (B1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (B1);
  • amino acid sequence of the light chain variable region is selected from:
  • the amino acid sequence shown in (B4) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (B4);
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention, wherein:
  • the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 110, 111, 112, 113 or 114, and SEQ ID NO: 110, 111, 112, 113 or 114 is substituted, deleted or added.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention, wherein:
  • the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 119, 120, 121, 122, or 123, and SEQ ID NO: 119, 120, 121, 122, or 123 is substituted, deleted or added.
  • the amino acid sequence of NO: 124, 125 or 126 is functionally identical or the amino acid sequence of SEQ ID NO: 124, 125 or 126 has at least 85% sequence identity.
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 112, and SEQ ID NO: 112 is The amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 112 or having at least 85%, or at least 90%, or at least 95%, or at least 98% with SEQ ID NO: 112 Sequence identity and the H2CDR1, H2CDR2 and H2CDR3 are as shown in SEQ ID NO: 75, 76 and 77, and the amino acid sequence of the light chain variable region is SEQ ID NO: 116, SEQ ID NO: 116 The amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 116 or having at least 85%, or at least 90%, or at least 95%, or at least 98 with SEQ ID NO: 116 % Sequence
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 123, and SEQ ID NO: 123 is The amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 123 or having at least 85%, or at least 90%, or at least 95%, or at least 98% with SEQ ID NO: 123 Sequence identity and the H2CDR1, H2CDR2, and H2CDR3 have the amino acid sequences shown in SEQ ID NOs: 87, 88 and 89, and the amino acid sequence of the light chain variable region is SEQ ID NO: 126, SEQ ID NO: 126 The amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 126 or having at least 85%, or at least 90%, or at least 95%, or at least 98 as SEQ ID NO
  • the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present invention wherein the antibody is a humanized antibody or a fully human antibody.
  • Another aspect of the invention provides isolated nucleic acids.
  • the isolated nucleic acid according to the invention encodes an anti-CD47/anti-PD-L1 antibody or an anti-PD-L1 antibody of the invention.
  • the isolated nucleic acid according to the invention encodes an anti-CD47 antibody or antigen-binding fragment thereof of the invention.
  • the isolated nucleic acid according to the invention encodes the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention.
  • the nucleotide sequence encoding the first heavy chain variable region SEQ ID NO: 30 is shown in SEQ ID NO: 36, and the nucleotide sequence encoding the first light chain can be The nucleotide sequence of the variable region SEQ ID NO: 33 is shown in SEQ ID NO: 39.
  • the nucleotide sequence encoding the second heavy chain variable region SEQ ID NO: 112 is shown in SEQ ID NO: 127 and encodes the second light chain
  • the nucleotide sequence of the variable region SEQ ID NO: 116 is shown in SEQ ID NO: 129.
  • the nucleotide sequence encoding the second heavy chain variable region SEQ ID NO: 123 is shown in SEQ ID NO: 128 and encodes the second light chain
  • the nucleotide sequence of the variable region SEQ ID NO: 126 is shown in SEQ ID NO: 130.
  • Another aspect of the invention provides an expression vector.
  • the expression vector of the present invention expresses the anti-CD47/anti-PD-L1 bispecific antibody or anti-PD-L1 antibody of the present invention.
  • the expression vector of the present invention expresses the anti-CD47 antibody or antigen-binding fragment thereof of the present invention.
  • the expression vector of the present invention expresses the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention.
  • the vector expressing the anti-CD47 antibody or the antigen-binding fragment thereof of the present invention and the vector expressing the anti-PD-L1 antibody or the antigen-binding fragment thereof of the present invention are the same kind of expression vector.
  • the expression vector according to the present invention contains the isolated nucleic acid molecule of the present invention.
  • Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
  • the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells.
  • the host cell is bacteria, preferably Escherichia coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method for preparing the anti-CD47/anti-PD-L1 bispecific antibody or anti-PD-L1 antibody of the present invention, comprising expressing the antibody in the host cell and isolating the antibody from the host cell step.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the anti-CD47/anti-PD-L1 bispecific antibody of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising the anti-CD47/anti-PD-L1 bispecific antibody of the present invention and other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of liposomes.
  • a chimeric antigen receptor (CAR) fusion protein comprising the anti-CD47 antibody or antigen-binding fragment thereof and/or anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention.
  • the chimeric receptor antigen fusion protein comprises an anti-CD47 antibody of the invention or antigen-binding fragment thereof, which variable fragment (scFv) of V H and V L, for a single chain CD47 antigen.
  • the chimeric antigen receptor fusion protein comprises the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention, which is a single-chain variable fragment of V H and V L against the PD-L1 antigen (scFv).
  • the chimeric receptor antigen fusion protein comprises an antigen against CD47 V H and V L of the first single-chain variable fragment (scFv) and L for PD-L1 antigen V H and V
  • L1CDR1 H1CDR1 first heavy chain variable region having the above described embodiments for the V H and V L, of the first scFv CD47 antigen, H1CDR2 H1CDR3 and a first and a light chain variable region, L1CDR2 and L1CDR3.
  • L2CDR1 H2CDR1 second heavy chain variable region having the above described embodiments of PD-L1 antigen for the V H and V L, the second scFv, H2CDR2 and H2CDR3 and second light chain variable region, L2CDR2 and L2CDR3.
  • the anti-CD47/anti-PD-L1 bispecific antibody of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration.
  • Administration methods include, but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes.
  • the formulation can be administered by any route, for example, by infusion or bolus injection, or by absorption through epithelial or mucosal skin (for example, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the preparation can be prepared by a method known in the art, and contains a carrier, diluent or excipient conventionally used in the field of pharmaceutical preparations.
  • Another aspect of the present invention provides a method of treating and/or preventing diseases related to CD47, PD-L1, or both, the method comprising administering the anti-CD47/anti-PD-L1 bispecific of the present invention to an individual in need thereof Sex antibody or the pharmaceutical composition of the present invention.
  • Another aspect of the present invention provides the anti-CD47/anti-PD-L1 bispecific antibody or anti-PD-L1 antibody of the present invention or the pharmaceutical composition of the present invention for preparing treatment and/or prevention with CD47, PD-L1 or both Application of drugs for related diseases.
  • the diseases associated with CD47, PD-L1, or both include hematological tumors, lymphomas, breast cancer, lung cancer, gastric cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder Cancer, pancreatic cancer, glioma and/or melanoma.
  • the tumor can be any tumor that expresses PD-L1 protein, such as bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer) Etc.), breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, etc.
  • the tumor can be a primary or metastatic tumor.
  • the present invention provides the use of the above-mentioned anti-CD47/anti-PD-L1 bispecific antibody or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs, for example, the tumor is selected from the group consisting of hematological tumors, lymphomas, Breast cancer, lung cancer, stomach cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.
  • the anti-CD47/anti-PD-L1 bispecific antibody provided by the present invention has a significant anti-tumor effect, can significantly inhibit tumor growth, and does not have obvious red blood cell toxicity.
  • the humanized antibody immunogenicity is greatly reduced and effectively eliminated
  • the rejection of exogenous monoclonal antibodies by the human immune system can be used in the preparation of drugs for the treatment of various tumor diseases, and has broad market prospects.
  • the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. %. Sequence comparison and determination of the percent identity between the two sequences can be performed by the BLASTN/BLASTP algorithm on the National Center For Biotechnology Instutute website.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in a three-dimensional space to form an antigen-binding surface.
  • the antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are called "complementarity determining regions" or "CDRs".
  • CDRs complementarity determining regions
  • the “antibody” of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • the antibody can be a whole antibody and any antigen-binding fragment or single chain thereof.
  • the "antibody” of the present invention includes any protein or peptide containing at least a part of the biological activity of binding an antigen that contains an Ig molecule. Examples of the “antibody” of the present invention include, but are not limited to, the CDR of a heavy chain or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region or any part thereof.
  • the "antigen-binding fragment" of the present invention includes Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human CD47 or PD-L1 that have antigen-binding activity.
  • the Fv fragment contains the variable region of the first heavy chain of the antibody and the variable region of the first light chain, but has no constant region and is the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • scFv single-chain antibody or single-chain Fv
  • the anti-CD47 or anti-PD-L1 antibody of the present invention may be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind to an antigen.
  • scFv include antibody polypeptides formed by recombinant DNA technology in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are connected via a spacer sequence.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • the antibody in the present invention refers to an immunoglobulin molecule or an immunologically active part thereof, that is, a molecule that contains an antigen binding site that specifically binds to (immunely reacts with) an antigen.
  • "Specific binding” means that an antibody reacts with one or more epitopes of an antigen but does not react with other polypeptides or binds to other polypeptides with very low affinity (Kd>10 -6 ).
  • Antibodies include but are not limited to polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv and Fab expression libraries.
  • Monoclonal antibody is an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, and synthesis technology such as CDR grafting or other existing technologies.
  • the "murine antibody” of the present invention is a monoclonal antibody against human CD47 prepared according to the knowledge and skills in the field. During preparation, the test subject is injected with CD47 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the "chimeric antibody” of the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed.
  • the gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the "humanized antibody” of the present invention is also called a CDR grafted antibody, and is an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework (FR).
  • Such variable region framework sequences can be obtained from public DNA databases or public references, for example, from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the "bispecific antibody” in the present invention refers to a monoclonal antibody having binding specificities for at least two different antigens.
  • the "peptide linker” of the present invention may be those including 1 to 10, especially 2 to 50, any amino acids, and may include any kind of amino acids without any limitation.
  • Figure 1 is the result of ELISA for the binding activity of anti-CD47 humanized antibody to monkey CD47.
  • Figure 2 shows the results of ELISA for the binding activity of anti-CD47 humanized antibodies to human CD47.
  • Figure 3 shows the results of ELISA for the binding activity of anti-CD47 humanized antibodies to cell surface CD47.
  • Figure 4 shows the results of red blood cell agglutination experiments, in which RBC: positive control; PBS: blank control.
  • Figure 5 is the FACS test results of the anti-CD47 humanized antibody blocking activity.
  • Figure 6 is the anti-tumor test result of the humanized anti-CD47 antibody Hu34-39-PE human gastric cancer NUGC-4 transplanted tumor model.
  • Figure 7 is the anti-tumor test result of the humanized anti-CD47 antibody Hu26T-31-PE human gastric cancer NUGC-4 transplanted tumor model.
  • Figure 8 is a schematic diagram of the structure of the double antibody ScFab (HuPL7-21Ks/Hu34-39Hs).
  • Figure 9 is a schematic diagram of the sequence of ScFabHuPL7-21Ks.
  • Figure 10 is a schematic diagram of the sequence of ScFabHu34-39Hs.
  • Figure 11 is the double antibody ScFab (HuPL7-21Ks/Hu34-39Hs), ScFab (HuPL16-42Ks/Hu34-39Hs) binding to PD-L1 (A), competing with PD-1 (B), and binding to CD47 (C) , Competition with SIRP ⁇ (D) and CD80 (E) experimental results.
  • Figure 12 shows the binding of scFab (HuPL7-21Ks/Hu34-39Hs), ScFab (HuPL16-42Ks/Hu34-39Hs) to cell surface PD-L1 (A), binding to cell surface CD47 (B), and blocking at the cellular level.
  • Figure 13 shows the CD47/PD-L1 double binding (A, B) and CD47/CD47/PD-L1 double binding (A, B) and CD47/ScFab (HuPL16-42Ks/Hu34-39Hs) on Raji-hPD-L1 cells.
  • Figure 14 is the experimental results of double anti-ScFab (HuPL7-21Ks/Hu34-39Hs) inhibiting the growth of transplanted tumors in mice.
  • the gene fragment encoding the full length of CD47 protein was synthesized, and the amino acid sequence design was as shown in SEQ ID NO: 41, and then cloned into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid pTargeT-CD47.
  • the amino acid sequence of the extracellular region of the human CD47 protein is fused with the amino acid sequence of hIgG1-Fc or his tag.
  • the design of the amino acid sequence is shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.
  • the labeled CD47 protein extracellular region gene fragments CD47-hFc and CD47-his were synthesized and cloned into the eukaryotic expression plasmid pHR to obtain the expression plasmid pHR-CD47- hFc, pHR-CD47-his.
  • the amino acid sequence of the extracellular region of human CD47 protein is fused with the amino acid sequence of mIgG1-Fc, and the amino acid sequence design is shown in SEQ ID NO: 44. After codon optimization of the amino acid sequence, a complete expression plasmid pcDNA3.1(+)-TPA-CD47-mIgG1-Fc was synthesized.
  • SIRP ⁇ The sequence of SIRP ⁇ is shown in SEQ ID NO: 45, and the complete expression plasmid pcDNA3.1(+)-SIRP ⁇ -myc-His is synthesized after codon optimization of the sequence.
  • AB06.12-4P Using the antibody AB6.12-IgG4P (abbreviated as AB06.12-4P herein) disclosed in the patent application WO2013/119714 as a positive control antibody, the amino acid sequence of AB06.12-4P is as follows:
  • the amino acid sequence corresponding to the above antibody sequence was artificially optimized to obtain the heavy and light chain expression plasmids pcDNA3.1(+)-SHC025-hG4, pcDNA3.1(+)- of the positive control antibody AB06.12-4P SHC025-hk.
  • the heavy chain gene fragment was cloned into the eukaryotic expression plasmid pHR containing the constant region of the IgG4 light chain, and the heavy chain eukaryotic expression plasmid pHR-SHC025-hG4-4PE of AB06.12-4P was obtained, and the light chain expression plasmid was pcDNA3.1(+)-SHC025-hk.
  • the eukaryotic expression plasmid pTargeT-CD47 was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) under a square pulse of 15msec at a voltage of 160V and placed in an incubator at 37°C and 5% CO 2 In the cultivation. After 24h, pressurized culture with 500ug/ml G418 medium. After 16 days, FACS was used to detect the positive rate of pool. The cells after electrotransformation were plated (1x10 6 cells/ml cell density, 100ul/well), and PE mouse anti-human CD47 antibody (BD, 556046) was used to incubate with the cells.
  • a cytometer (BD, FACSJazz) reads the mean value at a wavelength of 585nm, and uses GraphPad for data analysis.
  • the positive cell line was subcloned, and a cloned CHO-K1 cell line was selected, which expresses CD47 molecules at a high level and was named CHO-K1-E5.
  • the mixed solution of PEI and FreeStyle 293 expression medium was added to the plasmid, mixed evenly, and then added to the cell culture, placed in a 37°C, 8% CO 2 , humidified CO 2 incubator for cultivation.
  • the cells were fed on the 1st and 3rd day after cell transfection, 2.5ml of glutamine (mother liquor concentration of 200mM) and 5ml of glucose (mother liquor concentration of 180g/L) were added to each bottle. When the cell viability drops to 65%-75%, the cell supernatant is collected.
  • the cell culture was centrifuged at 1500 rpm for 5 min, and the supernatant was collected, and then centrifuged at 8000 rpm for 20 min, and the supernatant was collected.
  • Sample loading sample the cell expression supernatant, retention time 5min;
  • the collection starts when UV280 reaches about 50mAu, and stops when it drops to about 50mAu. Adjust the pH of the sample to 7.0 with 1M Tris-HCl, pH 9.0;
  • On-line cleaning 0.1M NaOH cleaning for 30min, retention time 5min;
  • mice of the SJL strain were immunized with different labels of anti-CD47 antigen protein and adjuvant.
  • the first antigen was 50ug antigen, and the latter was immunized with 25ug antigen.
  • the immune adjuvant can be Quick Antibody-Mouse5W (Beijing Boaolong Immunology Technology Co., Ltd.) or Titer Max (Sigma) and CpG (GenScript Biotechnology Limited Synthesis)/Alum (thermo) adjuvant interval. Add CD47 antigen protein samples with different labels to the adjuvant solution drop by drop, and vortex while dripping to mix thoroughly. Refer to the instructions for the dosage of the adjuvant. SJL mice were immunized after mixing uniformly to form a water-in-oil emulsion.
  • Cell lines expressing high levels of CD47 molecules such as CCRF-CEM and CHO-K1-E5 are also used to immunize mice to produce antibodies. Treat the human acute lymphocytic leukemia cells (CCRF-CEM) in culture and the CHO-K1-E5 positive single cells obtained in Example 1 with trypsin, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet in PBS, and sample Count with a cell counter, centrifuge the remaining sample at 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet in PBS, and add an appropriate amount of PBS to obtain a cell suspension of 1 ⁇ 10 8 cells/ml. Each experimental group of mice immunized 1x10 7 cells.
  • CCRF-CEM human acute lymphocytic leukemia cells
  • the immunization program is shown in Table 2:
  • the acquisition and preparation of spleen cells the boosted immunized mice were sacrificed and soaked in 75% alcohol. The spleen was dissected and taken out, ground with a grinding rod, and filtered through a cell sieve to prepare a single cell suspension. Centrifuge the spleen cell suspension at 2000 rpm for 5 min, and discard the supernatant. Add 2 mL of red blood cell lysis solution, lyse the red blood cells at room temperature for 2 minutes, add PBS to 20 mL, centrifuge at 1500 rpm for 7 minutes, discard the supernatant, and count the viable cells after resuspension.
  • the cell suspension was transferred to 15 mL RPMI1640 complete medium containing 20% FBS, and placed at room temperature for 20 minutes.
  • the fused cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, and 20% FBS.
  • Add the cell suspension to several 96-well cell culture plates at a rate of 100 ⁇ l/well to ensure that the cell volume per well is approximately 4 ⁇ 10 4 cells/well, and place them in a 37°C cell culture incubator. After 5 days, 100 ⁇ L/well of RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was added.
  • Coated SIRP ⁇ -myc-his on the ELISA plate add the recombinant human protein CD47-hFc and hybridoma supernatant mixture and incubate for 2h, add HRP-labeled antihumanIgG Fc specific antibody (Jackson ImmunoResearch) and incubate for 1h , Use a microplate reader to detect the absorbance at 450nm.
  • the hybridoma parent clones with binding ability and blocking ability obtained by screening are expanded and cultured, the binding activity and blocking activity are retested, and the hybridoma positive clones with binding and blocking ability are obtained by screening again.
  • the positive cell lines were subcloned by the limiting dilution method, and after one week of culture, the binding activity of the subclonal supernatant with the CD47 molecule and the activity of blocking the CD47-SIRP ⁇ interaction were detected by ELISA, and three double positive cell lines were obtained, which were labeled as SHC025-26, SHC025-34, SHC025-58.
  • the parent clones of monoclonal antibodies SHC025-26, SHC025-34, and SHC025-58 were determined and expanded.
  • the culture condition is 1640 medium containing 10% fetal bovine serum, 1x NAEE, 1x sodium pyruvate, and 1% penicillin double antibody.
  • the cell confluence is greater than >80%, the cells are subcultured and cultured. Collect the supernatant when it reaches about 50ml, and purify the antibody.
  • the obtained antibody was confirmed to be of good purity by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured, and the appropriate amount of cells was extracted according to the RNeasy Plus Mini Kit (Qiagen, 74134) kit instructions to extract total RNA, and Prime Script1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription reagent
  • the cassette synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the mouse antibody subtype (the 5'end contains the homologous arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as the template for PCR amplification of the antibody variable region gene , So as to obtain the gene fragments of the variable region of the mouse antibody light chain and heavy chain respectively; design primers (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester etal. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55; 2.
  • Antibody Amino acid sequence of heavy chain variable region Light chain variable region amino acid sequence SHC025-26 SEQ ID NO: 21 SEQ ID NO: 24 SHC025-34 SEQ ID NO: 22 SEQ ID NO: 25 SHC025-58 SEQ ID NO: 23 SEQ ID NO: 26
  • the CDR sequence of the VH of the antibody SHC025-34 the sequence of CDR1, CDR2, and CDR3 are SEQ ID NO: 4, 5, 6, respectively;
  • the CDR sequence of VL the sequence of CDR1, CDR2, CDR3 are SEQ ID NO: 15, 16 respectively , 17.
  • the mouse antibody light chain and heavy chain variable region gene fragments were respectively co-transformed with the linearized eukaryotic expression plasmid containing the human antibody light chain or heavy chain constant region to E. coli DH5 ⁇
  • E. coli DH5 ⁇ For competent cells, spread the mixture evenly on the surface of the agar plate containing the corresponding antibiotics. After culturing in a constant temperature incubator at 37°C overnight, select several single colonies for DNA sequencing; label the chimeric antibodies with correct sequencing as SHC025- 26CHI, SHC025-34CHI, SHC025-58CHI.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing corresponding antibiotics, cultured with shaking at 37°C for more than 12 hours, and then the bacteria were collected for plasmid extraction to obtain chimeric antibody light chain and heavy chain expression plasmids. Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the chimeric antibody was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and then tested for purity, activity and affinity.
  • amino acid sequences of SHC025-26CHI and SHC025-58CHI were modified as follows: the C118 of the heavy chain of SHC025-26CHI was mutated to T and marked as SHC025-26CHI-T; the C56 of the light chain of SHC025-58CHI was mutated to A, labeled as SHC025-58CHI-A, use site-directed mutagenesis to construct mutant genes.
  • the results of the chimeric antibody sequencing are shown in Table 5.
  • the CDR sequence of the VH of the antibody SHC025-34CHI the sequence of CDR1, CDR2, and CDR3 are SEQ ID NO: 4, 5, 6, respectively;
  • the CDR sequence of VL the sequence of CDR1, CDR2, CDR3 are SEQ ID NO: 15, 16 respectively , 17.
  • SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T were selected for the transformation of humanized antibodies according to the results of the activity analysis of the chimeric antibody and the affinity KD value.
  • the humanization of antibodies is firstly performed by comparing with the mouse antibody sequence in the Immunity Gene Database (IMGT) to confirm the murine origin of the variable regions of SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T antibodies.
  • IMGT Immunity Gene Database
  • Germline after homology comparison, the FR regions of the heavy chain variable region sequence of the SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T antibodies are respectively compared with the human antibody germline genes IGHV1-8*01, IGHV3- 21*04 and IGHV1-2*02 are the most similar; the FR sequences of the variable region of the antibody light chain are the most similar to human antibody germline genes IGKV3-11*01, IGKV1-5*01 and IGKV4-1*01, respectively.
  • SHC025-34CHI/SHC025-58CHI-A antibody framework region sequence FR1-FR3 as a template, look for a full human framework with similar 3D structure but low immunogenicity in the human framework region library to replace SHC025-34CHI/SHC025-58CHI-A 3D modeling of the FR1-FR3 sequence, heavy chain/light chain full-length sequence and structural comparison analysis with the original antibody heavy chain/light chain sequence, considering the antigenicity and 3D structural similarity, the final selection of SHC025-34CHI 6 humanized heavy chain variable regions (see SEQ ID NO: 48, 49, 50, 51, 52, 53) and 4 humanized light chain variable regions (see SEQ ID NO: 54, 55, 56 , 57) and 6 humanized heavy chain variable regions of SHC025-58CHI-A (see SEQ ID NO: 58, 59, 60, 61, 62, 63) and 5 humanized light chain variable regions ( See SEQ ID NO: 64, 65, 66, 67,
  • SHC025-34CHI/SHC025-58CHI-A humanized antibody non-CDR regions are more than 95% humanized.
  • 4 humanized heavy chain variable regions of SHC025-26CHI-T see SEQ ID NO: 69, 70, 71, 72
  • 2 humanized light chain variable regions see SEQ ID NO: 73 , 74).
  • the humanized antibody light chain and heavy chain variable region amino acid sequences designed above are reverse transcribed into corresponding nucleotide sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated by Overlap The oligonucleotide fragments are annealed and joined by PCR, and then specific primers (5' end containing homologous arm sequences for homologous recombination with eukaryotic expression vectors) are used to amplify the complete light and heavy chains.
  • Nucleotide fragments of the variable region; the purified nucleotide fragments of the variable region of the light chain and the linearized eukaryotic expression plasmid containing the constant region of the IgG4 light chain are co-transformed into E. coli DH5 ⁇ competent cells, and the purified heavy chain
  • the nucleotide fragments of the variable region and the eukaryotic expression plasmid containing the S228P/L235E mutant IgG4 heavy chain constant region were co-transformed into E. coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were evenly spread on the agar containing the corresponding antibiotics. After culturing the surface of the plate in a constant temperature incubator at 37°C overnight, several single colonies were picked for DNA sequencing.
  • Transfect the plasmid into HEK293E cells express and purify to obtain a large number of antibodies, and conduct purity testing, activity analysis and affinity testing.
  • the CDR sequence of the VH of the antibody Hu34-39-PE the sequence of CDR1, CDR2, and CDR3 are SEQ ID NO: 4, 5, 6, respectively;
  • the CDR sequence of VL the sequence of CDR1, CDR2, CDR3 are SEQ ID NO: 15 respectively , 16, 17.
  • Protein based Elisa was used to analyze the binding activity of antibodies. Cynomolgus CD47-His (0.1 ⁇ g/well, ACRO Biosystems, Cat. No. CD7-C52H1-50ug) was coated with a 96-well microtiter plate.
  • the anti-CD47 antibody provided by the present invention starts from 2 ⁇ g/mL as a primary antibody, and adds 5-fold gradient dilution to the ELISA plate.
  • a total of 8 concentrations are 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2 ng/mL, 0.64ng/mL, 0.128ng/mL, 0ng/mL, incubated at 37°C for 1.5h, the positive control antibody is AB06.12-4P.
  • Use Anti-Human IgG HRP Jackson, 109-035-003, 1:10000 as the secondary antibody, add color developing solution TMB (3,3',5,5'-tetramethylbenzidine), and use enzyme label after termination
  • An instrument thermo, Multiskan FC
  • Using GraphPad to generate EC 50 the result is shown in Figure 1.
  • ELISA was used to analyze the binding activity of antibodies.
  • Human CD47-His protein (0.1ug/well, prepared in Examples 1 and 2) was coated on a 96-well microtiter plate, and incubated at 37°C for 2h. After washing 3 times with 1xPBST, it was blocked with 5% skimmed milk at 4°C overnight. After washing 3 times with 1xPBST, the anti-CD47 antibody provided by the present invention is used as a primary antibody starting from 2 ⁇ g/mL, and added to the ELISA plate in 5-fold serial dilutions. A total of 8 concentrations are 2000ng/mL, 400ng/mL, and 80ng/mL.
  • the positive control antibody is AB06.12-4P; after washing 5 times with 1xPBST, the secondary antibody Using Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), incubate at 37°C for 40 min. After washing 5 times with 1xPBST, add the color developing solution TMB, and use the microplate reader (thermo, Multiskan FC) to read the OD450 value after termination. Using GraphPad to generate EC 50 , the result is shown in Figure 2.
  • CHO-K1-E5 cells were plated with 1 ⁇ 10 5 cells per well and cultured overnight at 37°C and 5% CO 2 ; fixed with 4% paraformaldehyde and blocked with skim milk the next day 1h; Wash the cell plate gently with 1xPBS; the anti-CD47 antibody provided by the present invention is used as a primary antibody starting from 2 ⁇ g/mL, and added to the cell plate in 5-fold dilutions.
  • a total of 8 concentrations are 2000ng/mL, 400ng/mL, and 80ng.
  • the positive control antibody is AB06.12-4P; the secondary antibody uses Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), add the color developing solution TMB, and read the OD450 value with a microplate reader (thermo, Multiskan FC) after termination.
  • GraphPad to generate EC 50 , the result is shown in Figure 3.
  • the experimental results show that the humanized anti-CD47 antibodies Hu26T-31-PE, Hu34-39-PE, Hu58A-14-PE provided by the present invention can bind to CD47 on the cell surface, and the binding ability is the same as that of the positive control antibody AB06.12- 4P is equivalent.
  • the CD47-hFc-Biotin was diluted with SD buffer (0.02% Tween20+0.1% BSA solution) to a concentration of 5ug/ml, and the humanized anti-CD47 antibody was diluted with SD buffer 4-fold concentration gradient to make the concentration 10ug /ml, 2.5ug/ml, 0.625ug/ml, 0ug/ml, select SA sensor to solidify the antigen, and perform affinity determination according to the operating procedures of Fortebio Octet RED96.
  • the specific parameters and experimental results are shown in Table 8.
  • the experimental results show that, compared with the positive control antibody, the humanized anti-CD47 antibody Hu26T-31-PE binds to human CD47 protein with higher affinity.
  • the initial concentration of the antibody to be analyzed is between 1-20uM, with a 2-fold gradient dilution, a total of 24 concentration gradients.
  • Rabbit polyclonal antibody RBC antibody (Rockland, 109-4139) was used as a positive control for hemagglutination, and the results are shown in Figure 4.
  • the concentration of the antibodies (rabbit polyclonal antibody RBC antibody, AB06.12-4P antibody and the test antibody of the present invention) added from left to right in the 96-well plate was diluted in a 2-fold gradient from 20uM, where RBC Indicates the positive control group (using the rabbit polyclonal antibody RBC antibody, which significantly causes agglutination of red blood cells), and PBS represents the blank control group.
  • the red blood cells in the hole are small dots, and the edges are neat, indicating that they do not cause cell agglutination; slightly irregular edges indicate a small amount of red blood cells agglutination; flaky, covering the bottom of the hole indicates that most of the red blood cells agglutination.
  • FACS is used to detect the ability of the anti-CD47 antibody provided by the present invention to block SIRPa from binding to CD47 on the cell surface.
  • the CHO-K1-E5 positive cell line is used as a CD47 provider.
  • the secondary antibody uses PE Streptavidin (Biolegend, 405203, 1:200) to monitor the changes of SIRPa-Biotin.
  • AB06.12-4P was used as a positive control to block SIRPa from binding to CD47 on the cell surface.
  • a flow cytometer (BD, FACSJazz) reads the mean value at a wavelength of 585 nm, and uses GraphPad to generate IC50. The result is shown in Figure 5.
  • the experimental results showed that the blocking activity was ranked as Hu26T-31-PE ⁇ Hu34-39-PE ⁇ AB06.12-P>Hu58A-14-PE.
  • NUGC-4 human gastric cancer cells were purchased from the American Type Culture Collection (ATCC);
  • NOD-Scid mice female, 5-8 weeks old, weighing 18-20 grams, purchased from Shanghai Lingchang Biological Technology Co., Ltd.;
  • the reference substance Isotype IgG4 (Cat. No. AB170091) was purchased from Sino-American Crown Biotechnology Co., Ltd. and used as a negative reference substance;
  • the humanized anti-CD47 antibody of the present invention was prepared with PBS at two concentrations of 0.6 mg/mL and 0.3 mg/mL, and Isotype IgG4 and AB 06.12-4P were prepared at 0.6 mg/mL.
  • the RMPI1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin was used to culture NUGC-4 human gastric cancer cells in an incubator at 37° C. and 5% CO 2. Digest with 2mL 1 ⁇ EDTA solution once a week for passage. When the cell saturation is 80%-90%, the cells are collected, counted, and seeded. Mix PBS containing 5 ⁇ 10 6 cells with 100 uL of Matrigel (final volume 200 ⁇ L) and inoculate the mice on the right back side. The number of inoculated cells is 5 ⁇ 10 6 /mouse.
  • T/C Relative tumor proliferation rate
  • RTV V21/V0, where V0 is the tumor volume measured at the time of group administration (i.e. d0), and V21 is the tumor volume measured at 21 days of administration.
  • the tumor volume on the last day (day21) of the administration group and the vehicle group was analyzed by T-test and performed by GraphPad Prism. The results are shown in Table 9.
  • the mFc-labeled PD-L1 antigen protein (purchased from Beijing Biosciences Biotechnology Co., Ltd.) was used to immunize BALB/c, SJL strains of experimental mice and SD strains of experimental rats by co-immunization with adjuvants.
  • the immune adjuvant was Freund’s Adjuvant, Complete (SIGMA, F5881-10ML) for the first time, and Freund’s Adjuvant, Incomplete (SIGMA, F5506-10ML) was used later.
  • the PD-L1 antigen protein samples of different labels were added dropwise to the adjuvant solution, and the adjuvant solution was vortexed while dripping to fully mix. The dosage of the adjuvant was carried out according to the instructions. Mice or rats are immunized after mixing uniformly to form a water-in-oil milk.
  • the immunization program is shown in Table 10:
  • the fused cell suspension was transferred to 15 mL RPMI 1640 complete medium containing 20% FBS, and placed at room temperature for 20 minutes.
  • the fused cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, and 20% FBS.
  • Add the cell suspension to several 96-well cell culture plates at a rate of 100 ⁇ l/well to ensure that the cell volume per well is approximately 4 ⁇ 10 4 cells/well, and place them in a 37°C cell culture incubator. After 5 days, 100 ⁇ L/well of RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was added.
  • the positive cell lines were subcloned by the limiting dilution method, and after one week of culture, the binding activity of the subclonal supernatant with PD-L1 molecules and the activity of blocking the interaction of PD-L1/PD-1 were detected by ELISA to obtain PL-7,
  • the four strains of PL-15, PL-16 and PL-18 are preferably double-positive cell strains.
  • the subcloned positive hybridoma cells were expanded and cultured, and the appropriate amount of cells was extracted according to the RNeasy Plus Mini Kit (Qiagen, 74134) kit instructions to extract the total RNA, and the PrimeScript 1st strand cDNA Synthesis Kit (Takara, 6110A) was used for reverse transcription.
  • the kit synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the mouse antibody subtype (the 5'end contains the homologous arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as the template for PCR amplification of the antibody variable region gene , So as to obtain the gene fragments of the variable region of the mouse antibody light chain and heavy chain respectively, named SHS009PL-7, SHS009PL-15, SHS009PL-16, SHS009PL-18; design primers (Reference: 1.
  • the purified mouse antibody light chain and heavy chain variable region gene fragments were respectively co-transformed with the linearized eukaryotic expression plasmid containing human antibody light chain or heavy chain constant region gene fragments to transform E. coli DH5 ⁇ competent cells, and picked
  • the chimeric antibodies that were sequenced correctly were labeled PL-7CHI, PL-15CHI, PL-16CHI, and PL-18CHI.
  • the sequencing results of the chimeric antibodies are shown in Table 12.
  • amino acid sequences of the light and heavy chain variable regions of PL-7CHI, PL-15CHI, PL-16CHI, and PL-18CHI are the same as those of the murine antibodies SHS009PL-7, SHS009PL-15, SHS009PL-16, and SHS009PL-18. The sequence is consistent.
  • the chimeric antibody light chain plasmid and heavy chain plasmid were extracted and transfected into HEK 293F cells, and a large number of antibodies were obtained by expression and purification, and the purity test, activity analysis and affinity test were performed.
  • PL-7CHI and PL-16CHI were selected for humanized antibody modification.
  • the humanization of murine monoclonal chimeric antibodies PL-7CHI and PL-16CHI was carried out according to the classic CDR transplantation strategy, using PL-7CHI and PL-16CHI antibody framework region sequences FR1-FR3 as templates, in the human framework region library Looking for a full human framework with similar 3D structure but low immunogenicity to replace the FR1-FR3 sequence of PL-7CHI/PL-16CHI.
  • the FR regions of the heavy chain variable regions of PL-7CHI and PL-16CHI antibodies are respectively matched with human antibody germline genes M99683
  • the humanized heavy chain/light chain full-length sequence is 3D modeling and structural comparison analysis with the original antibody heavy chain/light chain sequence, comprehensively considering antigenicity and 3D structural similarity, and will be displayed in the structure simulation
  • the amino acid sites that play a key role in the stability of the antibody structure are back mutated into murine amino acid residues.
  • PL-7CHI humanized heavy chains were obtained (PL-7CHI humanized heavy chain variable region sequence: VH1-0 (SEQ ID NO: 110), VH1-1 (SEQ ID NO: 111), VH1- 2 (SEQ ID NO: 112), VH1-3 (SEQ ID NO: 113) or VH1-4 (SEQ ID NO: 114)), 4 humanized light chains (PL-7CHI humanized light chain variable Region sequence: VL1-0 (SEQ ID NO: 115), VL1-1 (SEQ ID NO: 116), VL1-2 (SEQ ID NO: 117) or VL1-3 (SEQ ID NO: 118)); PL- 16CHI humanized heavy chain 5 (PL-16CHI humanized heavy chain variable region sequence: VH1-0 (SEQ ID NO: 119), VH1-1 (SEQ ID NO: 120), VH1-2 (SEQ ID NO: 121), VH1-3 (SEQ ID NO: 122) or VH1-4 (SEQ ID NO: 123)), 3 humanized light chains (PL-16CHI humanized light chain variable region sequence: VH1-0
  • the above-designed humanized antibody light chain and heavy chain variable region amino acid sequences are synthesized into corresponding nucleotide coding sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated. Oligonucleotide fragments are annealed and joined together, and then specific primers (5' end containing homologous arm sequences for homologous recombination with eukaryotic expression vectors) are used to amplify the complete light chain and heavy chain variable regions Nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid containing the IgG4 light chain constant region are co-transformed into E.
  • E. coli DH5 ⁇ competent cells and the purified heavy chain is variable Co-transformation of E. coli DH5 ⁇ competent cells with the eukaryotic expression plasmid containing the S228P/L235E mutant IgG4 heavy chain constant region, and the competent cells of the transformed plasmid are uniformly spread on the surface of the agar plate containing the corresponding antibiotics. After culturing overnight in a constant temperature incubator at 37°C, several single colonies were picked for DNA sequencing.
  • Plasmid extraction is performed on the positive clones with correct sequencing to obtain humanized antibody light chain and heavy chain expression plasmids, and the concentration and purity of the plasmids are detected by a nucleic acid quantitative analyzer.
  • the plasmid was transfected into HEK293F cells, expressed and purified to obtain a large number of antibodies, and then tested for purity, activity and affinity.
  • the sequence is shown in Table 13.
  • the CDR sequence of the VH of the antibody HuPL7-21 the sequence of CDR1, CDR2, and CDR3 are SEQ ID NO: 75, 76 and 77, respectively
  • the CDR sequence of VL the sequence of CDR1, CDR2, CDR3 are SEQ ID NO: 78, 79, respectively And 80.
  • the CDR sequence of the VH of the antibody HuPL16-42 the sequence of CDR1, CDR2, and CDR3 are SEQ ID NO: 87, 88 and 89, respectively
  • the CDR sequence of VL the sequence of CDR1, CDR2, CDR3 are SEQ ID NO: 90, 91, respectively And 92.
  • the anti-CD47/anti-PD-L1 bispecific antibody was constructed by genetic engineering methods.
  • the structure of the bispecific antibody is shown in Figure 8. It is formed by dimerization of the source; different from natural IgG antibodies, the light chains of the anti-PD-L1 and anti-CD47 antibodies in this bispecific antibody are both connected to the N-terminus of the heavy chain of the antibody by an additional flexible connecting peptide, which is The GGGGS repetitive sequence containing glycine (G) and serine (S) residues preferably contains 8 GGGGS repetitive sequences; in addition, in order to promote the formation of heterodimers, based on the above-mentioned S228P/L235E mutations, the anti- The CH3domain of the PD-L1 antibody single chain adds S354C/T366W mutation, and the CH3domain of the anti-CD47 antibody single chain adds Y349C/T366S/L368A/Y407V mutation.
  • the above-mentioned anti-PD-L1 antibody HuPL7-21 (light chain variable region SEQ ID NO: 116 and constant region CL SEQ ID NO: 131) or HuPL16-42 (light chain variable region SEQ ID NO: 126 and the constant region CL SEQ ID NO: 131) sequence the nucleotide fragment of the single chain of the anti-PD-L1 antibody is obtained through gene synthesis, namely ScFabHuPL7-21Ks or ScFabHuPL16-42Ks;
  • the above-mentioned anti-CD47 antibody Hu34-39 The light chain variable region SEQ ID NO: 33 and the constant region CL SEQ ID NO: 131) sequences, the nucleotide fragments of the single chain of the anti-CD47 antibody, namely ScFabHu34-39Hs, are obtained through gene synthesis.
  • HuPL7-21VL-CL humanized PD-L1 monoclonal antibody HuPL7-21 light chain
  • (GGGGS) 8 a flexible connecting peptide of 8 GGGGS repeats
  • HuPL7-21VH humanized PD-L1 monoclonal antibody HuPL7-21 heavy chain variable region
  • IgG4CH/Ks IgG4 heavy chain constant region containing S228P/L235E/S354C/T366W mutation to form "Knobs" structure (specific sequence such as SEQ ID NO: 132);
  • Hu34-39VL-CL humanized CD47 monoclonal antibody Hu34-39 light chain
  • (GGGGS)6 6 flexible connecting peptides of GGGGS repeat sequence
  • Hu34-39VH humanized CD47 monoclonal antibody Hu34-39 heavy chain variable region
  • IgG4CH/Hs Contains S228P/L235E/Y349C/T366S/L368A/Y407V mutations
  • the IgG4 heavy chain constant region of the "Hole” structure (see SEQ ID NO: 133 for the specific sequence).
  • the CL sequence in the above structure is shown in SEQ ID NO: 131.
  • the Expi-Fectamine CHO Transfection Kit plasmid transfection kit to transfect the two expression vectors of ScFabHuPL7-21Ks (or ScFabHuPL16-42Ks) and ScFabHu34-39Hs into Expi-CHO cells, and culture them in serum-free medium 14 After days, the Expi-CHO cell supernatant was collected, and the expression of the bispecific antibody was detected by Western blotting.
  • the bispecific antibody formed by the two single-chain dimerization of ScFabHuPL7-21Ks and ScFabHu34-39Hs is named ScFab (HuPL7-21Ks/Hu34-39Hs), which will be composed of ScFabHuPL16-42Ks and ScFabHu34-39Hs.
  • the bispecific antibody formed by polymerization was named ScFab (HuPL16-42Ks/Hu34-39Hs).
  • the bispecific antibody of the present invention is expressed and secreted in Expi-CHO cells, it is purified by Protein A affinity chromatography.
  • the specific method is as follows: After the Protein A affinity chromatography column is equilibrated with buffer, the The supernatant of Expi-CHO cell culture medium concentrated by the ultrafilter was injected and monitored with A280 (nm), washed with the washing solution until all unbound proteins were eluted, and then eluted with the eluent To obtain the corresponding bispecific antibody.
  • the purified bispecific antibody was tested for purity by SEC-HPLC, and molecular weight was tested by LC-MS. After quality identification, it was used for subsequent pharmaceutical research.
  • hPD-L1 human PD-L1
  • Raji cells from Immune Onco Biomedical Technology Co., Ltd.
  • the human PD-L1-His protein (0.5ug/ml, 100ul/well) was coated on a 96-well microtiter plate, and incubated at 37°C for 2h. Washed with 1xPBST 3 times, then blocked with 5% skim milk at 4°C overnight, and washed 3 times with 1xPBST. The concentration of the double antibody starts from 10 ⁇ g/mL, and the 5-fold dilution is added to the ELISA plate, and incubated at 37°C for 1.5h.
  • the control antibody is Atezolizumab (Sino Biological, Cat: 68049-H001, abbreviated as Ate); after washing 5 times with 1xPBST, add HRP-Anti-Human IgG secondary antibody (Jackson, 109-035-003, 1:10000), incubated at 37°C for 40 min. Wash 5 times with 1xPBST, add color developing solution TMB, read the OD450 value with a microplate reader (thermo, Multiskan FC) after termination, and the EC 50 result is shown in Figure 11.
  • ELISA was used to analyze the activity of antibodies blocking the binding of PD1 and PD-L1.
  • the human PD-L1-hFC protein (2ug/ml, 100ul/well) was coated on a 96-well microtiter plate, and incubated at 37°C for 2h. After washing 3 times with 1xPBST, it was blocked with 5% skimmed milk at 4°C overnight.
  • the control antibody is Atezolizumab; after washing 5 times with 1xPBST, the second Anti-Mouse IgG HRP (Jackson, 109-035-003, 1:10000) was used to incubate at 37°C for 1 hour. After washing 5 times with 1xPBST, add the color developing solution TMB, and use the microplate reader to read the OD450 value after termination. The result is shown in Figure 11.
  • ELISA was used to analyze the binding activity of antibodies to CD47.
  • the human CD47-His protein (0.5ug/ml, 100ul/well) was coated on a 96-well microtiter plate, and incubated at 37°C for 2h. After washing 3 times with 1xPBST, it was blocked with 5% skimmed milk at 4°C overnight.
  • the concentration of the anti-CD47/anti-PD-L1 double antibody provided by the present invention starts from 50 ⁇ g/mL, and the 5-fold dilution is added to the ELISA plate, a total of 8 concentrations, and incubated at 37°C for 1.5h, the control antibody is Hu34-39-PE: After washing 5 times with 1xPBST, use HRP-Anti-Human IgG (1:10000) as the secondary antibody and incubate at 37°C for 40 minutes. After washing 5 times with 1xPBST, add the color developing solution TMB, and use the microplate reader (thermo, Multiskan FC) to read the OD450 value after termination. The EC 50 results are shown in Figure 11.
  • the experimental results show that the bispecific antibodies ScFab (HuPL7-21Ks/Hu34-39Hs) and ScFab (HuPL16-42Ks/Hu34-39Hs) have the ability to bind to human CD47, and the binding activity is reduced to varying degrees. ScFab (HuPL7 -21Ks / Hu34-39Hs) binding than 50 EC 50 values of EC value Hu34-39-PE 40 times.
  • ELISA was used to analyze the blocking activity of antibodies.
  • the human CD47-His protein (0.4ug/ml, 100ul/well) was coated on a 96-well microtiter plate, and incubated at 37°C for 2h. After washing 3 times with 1xPBST, it was blocked with 5% skimmed milk at 4°C overnight.
  • ELISA was used to analyze the activity of antibodies blocking the binding of PD-L1 to CD80.
  • the CD80-hFc protein (8ug/ml, 100ul/well) was coated on a 96-well microtiter plate and incubated overnight at 4°C. After washing 3 times with 1xPBST, it was blocked with 5% skimmed milk at 37°C for 2h.
  • the above experimental results show that the double antibody of the present invention can differentially bind to PD-L1/CD47, thereby ensuring anti-tumor activity while reducing the toxicity of the antibody, such as blood toxicity.
  • CHO-K1-hPD-L1/CHO-K1-hCD47 stably transfected cell line serves as the PD-L1/CD47 provider, and the gradiently diluted anti-PD-L1/anti-CD47 double antibody is added to the cell plate as the primary antibody, and incubated at 4°C for 1.5 h.
  • Atezolizumab and Hu34-39-PE were used as positive controls.
  • the flow cytometer reads the product of the mean value and the parent value at a wavelength of 585 nm, and the result is shown in Figure 12.
  • the experimental results show that the bispecific antibodies ScFab (HuPL7-21Ks/Hu34-39Hs) and ScFab (HuPL16-42Ks/Hu34-39Hs) have the ability to bind human PD-L1 on the cell surface, and the binding ability is equivalent to Atezolizumab.
  • the binding activity of double antibodies ScFab (HuPL7-21Ks/Hu34-39Hs) and ScFab (HuPL16-42Ks/Hu34-39Hs) to cell surface CD47 is lower than that of Hu34-39-PE monoclonal antibody, and the EC 50 is about 4 times higher, Emax Decrease about 2 times.
  • CHO-K1-hPD-L1/CHO-K1-hCD47 stably transfected cell line as a PD-L1/CD47 provider, observe PD-L1 and PD in the presence of the anti-PD-L1 antibody/anti-CD47 antibody in a gradient dilution -1 binding, CD47 and SIRP ⁇ binding ability.
  • the bispecific antibody ScFab HumanPL7-21Ks/Hu34-39Hs
  • ScFab Human-42Ks/Hu34-39Hs
  • Atezolizumab was used as a positive control for blocking the binding of PD-1-mFc to PD-L1 on the cell surface
  • Hu34-39-PE was used as a positive reference for blocking the binding of SIRP ⁇ to CD47 on the cell surface. The results are shown in Figure 12.
  • the experimental results show that the bispecific antibodies ScFab (HuPL7-21Ks/Hu34-39Hs) and ScFab (HuPL16-42Ks/Hu34-39Hs) can block human PD-1 and CHO-K1-PD-L1, SIRP ⁇ and CHO-
  • the ability of K1-CD47 to bind and the ability to block the binding of PD-1 to CHO-K1-PD-L1 is equivalent to Atezolizumab.
  • Hu34-39-PE the ability of the double antibody to block the binding of SIRP ⁇ and CHO-K1-CD47 was reduced, and the IC 50 value increased by 3 times.
  • the binding and blocking ability of double antibody molecules ScFab (HuPL7-21Ks/Hu34-39Hs), ScFab (HuPL16-42Ks/Hu34-39Hs) and PD-L1 is consistent with that of Atezolizumab.
  • the binding ability of the double antibody molecule with CD47 has decreased.
  • ELISA assay EC 50 increased about 40-fold, about 4-fold increase in 50 FACS analysis EC, Emax 2-fold decrease.
  • the double antibody molecules ScFab (HuPL7-21Ks/Hu34-39Hs) and ScFab (HuPL16-42Ks/Hu34-39Hs) differentially bind PD-L1/CD47, which helps to enhance the tumor targeting of the double antibody and reduce the double antibody The adverse reactions, especially for the adverse reactions of red blood cells.
  • Raji-hPD-L1 tumor cells were purchased from Immune Onco Biomedical Technology Co., Ltd.
  • hCD47 is also highly expressed.
  • the bispecific antibody ScFab HumanPL7-21Ks/ Hu34-39Hs
  • ScFab HumanPL16-42Ks/Hu34-39Hs
  • the binding of L1 shows the activity of double-arm binding and double-arm blocking.
  • Double-arm blocking Pave 2.4 ⁇ 10 5 Raji-hPD-L1 cells per well on the bottom of the U-shaped plate, add primary antibody, and the working concentration of the antibody starts from 2.5 ⁇ g/mL.
  • SIRP ⁇ -mFc with a concentration of 1 ⁇ g/mL and PD-1-mFc with a final concentration of 1 ⁇ g/mL are uniformly mixed.
  • 50 ⁇ l of antibody and 50 ⁇ l of antigen are premixed per well, and a total of 100 ⁇ l is added to the cell well.
  • NSG mice have the characteristics of NOD, Prkdcscid and IL2rgnull deletion/variation. They are currently the most immunodeficient and the most suitable tool for human cell transplantation. They have almost no rejection of human cells and tissues.
  • the present invention uses NSG mice (purchased from Beijing Biocytogene Biotechnology Co., Ltd.), Raji-hPD-L1 tumor cells to establish a tumor transplantation model-Raji-PBMC-NSG model to study anti-CD47/anti-PD-L1 double antibodies
  • the anti-tumor effect of ScFab HumanPL7-21Ks/Hu34-39Hs
  • CD47 monoclonal antibody 5F9 was purchased from Sino Biological Inc. (Cat: 68063-H001).
  • Table 14 is the experimental design plan for the anti-tumor effect of the tested drugs in the Raji-PBMC-NSG tumor model.
  • N refers to the number of mice in each group
  • BiAb refers to the double antibody ScFab (HuPL7-21Ks/Hu34-39Hs)
  • the changes of tumor volume in each group over time are shown in Figure 14.
  • the anti-tumor effect of the double antibody of the present invention is significantly better than that of Atezolizumab and better than the combination of monoclonal antibodies (Atezolizumab+5F9).
  • Example 20 Acute toxicity test of anti-CD47/anti-PD-L1 bispecific antibody cynomolgus monkey
  • This example is a toxicity test of the anti-CD47/anti-PD-L1 bispecific antibody ScFab (HuPL7-21Ks/Hu34-39Hs) (BiAb for short) administered to cynomolgus monkeys by a single intravenous infusion.
  • the dosage of the double antibody was 10, 30, 100 mg/kg, and each group of cynomolgus monkeys had one male and one male.
  • the dose of anti-CD47 monoclonal antibody Hu34-39-PE was 30 mg/kg, and the dose of Hu5F9 was 20 mg/kg, with 2 cynomolgus monkeys in each group.
  • a single intravenous infusion the observation period is 21 days. Blood is collected from the femoral vein at different time points for the detection of blood cell counts, coagulation function indexes, blood biochemical indexes, etc.
  • the anti-CD47/anti-PD-L1 bispecific antibody molecule ScFab (HuPL7-21Ks/Hu34-39Hs) of the present invention differentially binds CD47 and PD-L1, and better retains the binding to PD-L1 And blocking activity, at the same time, there is no red blood cell toxicity and other hematological toxicity, and the safety is excellent.
  • the present invention uses NSG mice (purchased from Beijing Biocytogene Biotechnology Co., Ltd., China), Raji-PD-L1 tumor cells (purchased from Imming Onco Biomedical Technology Co., Ltd., China) to establish a tumor transplantation model-Raji -PBMC-NSG model to study the anti-tumor effect of the antibody of the present invention in Raji-hPD-L1 lymphoma subcutaneous transplantation model.
  • the anti-PD-L1 positive control antibody is Atezolizumab (Sino Biological, Cat: 68049-H001). Each group has 6 mice.
  • the negative control group was given physiological saline (PBS).
  • the dosages of the anti-PD-L1 antibodies HuPL7-21 and Atezolizumab of the present invention are 10 mg/kg, respectively.
  • the route of administration is intraperitoneal injection, and the frequency of administration is 2 times a week for 3 consecutive weeks.
  • TGI TV tumor inhibition rate
  • the anti-PD-L1 antibody HuPL7-21 of the present invention has an anti-tumor effect in vivo that is significantly better than that of Atezolizumab.

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Abstract

提供了一种抗CD47/抗PD-L1抗体，以及抗CD47/抗PD-L1抗体的药物组合物及其应用。本发明的抗CD47/抗PD-L1抗体具有抗肿瘤活性，且不具有明显的红细胞毒性，可在制备抗肿瘤的药物中应用。

Description

抗CD47/抗PD-L1抗体及其应用 技术领域
本发明涉及抗体药物技术领域，尤其涉及抗CD47/抗PD-L1抗体，包含抗CD47/抗PD-L1抗体的药物组合物及其应用。
背景技术
CD47蛋白又称整合素相关蛋白(IAP)，属于IgG超家族的一个五次跨膜糖蛋白，广泛表达于不同组织细胞。CD47可与配体TSP-1或SIRPα结合，调控不同的细胞功能，包括细胞迁移、粘附、凋亡，轴突延伸，细胞因子产生及T细胞激活。SIRPα是一个含有典型免疫受体酪氨酸抑制基序(ITIM)跨膜蛋白，主要表达于髓系造血细胞膜表面，如巨噬细胞，树突状细胞等。CD47与SIRPα结合后，导致ITIMs的磷酸化，从而招募SHP-1/SHP-2，进而抑制肌球蛋白IIA在吞噬突触的积累，最终抑制吞噬细胞的吞噬功能。
肿瘤细胞的“免疫逃逸”被认为是肿瘤发生、发展和抗药的主要机制。肿瘤细胞通过高表达CD47分子，与巨噬细胞表面的SIRPα相互作用，可显著抑制巨噬细胞的吞噬活性，避免被巨噬细胞吞噬掉。当阻断CD47与SIRPα的结合时，可消除因肿瘤导致的免疫抑制或免疫耐受，有效杀伤肿瘤细胞。这为CD47的肿瘤免疫靶向治疗提供了极为有力的理论依据。
近年来，国内外针对CD47/SIRPα信号通路的各种治疗方式进行了大量研究，其中CD47阻断性抗体被认为是其中最有希望的肿瘤治疗方案。人CD47阻断型单抗的有效性已经在多种临床前模型中证实。不过，由于红细胞、血小板也表达CD47分子，当抗体阻断CD47与SIRPα的相互作用时，可能导致这些细胞失去“别吃我”信号的保护，进而被巨噬细胞吞噬。因此要避免抗CD47抗体的副作用，比如血小板降解，红细胞凝集，红细胞耗竭，贫血等也是应用抗CD47抗体需要考虑的一个重点。
程序性死亡配体1(PD-L1)，也称为分化簇274(CD274)或B7同系物1(B7-H1)，是一种40kDa的1型跨膜蛋白，其在特定事件(例如妊娠、组织同种异体移植、自身免疫疾病和其他疾病状态如肝炎)期间在抑制免疫***的过程中起主要作用。PD-L1与PD-1或B7.1的结合传递抑制性信号，该抑制性信号减少 CD8 +T细胞在***处的增殖，并且对该PD-1的补充也能够通过进一步由基因Bcl-2的更低调节介导的细胞凋亡来控制外来抗原特异性T细胞在***中的积累。
已经显示PD-L1的上调可以允许癌症逃避宿主免疫***。对来自肾细胞癌患者的肿瘤样本的分析发现，PD-L1的高肿瘤表达与增加的肿瘤侵袭性和增加的死亡风险相关。许多PD-L1抑制剂作为免疫肿瘤学疗法正在开发中，并且在临床试验中正在展现出良好的结果。
CD47-SIRPα信号通路不但激活固有免疫，巨噬细胞还将肿瘤抗原呈递给CD8+T、CD4+T细胞，通过促进T细胞激活，进一步杀伤肿瘤。因此，可以开发一种靶向CD47与PD-L1的双功能融合蛋白，该融合蛋白包含CD47结合部分和PD-L1结合部分，既能阻断PD-L1与PD-1的结合，又能阻断CD47与SIRPα的结合，把先天免疫和获得性免疫信号通路桥联在一起，从而具有更好的抗肿瘤活性、肿瘤靶向性和更低的红细胞毒性。
发明内容
本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段包含第一重链可变区和/或第一轻链可变区，其中所述第一重链可变区包含第一重链可变区的互补决定区1(H1CDR1)、第一重链可变区的互补决定区2(H1CDR2)和/或第一重链可变区的互补决定区3(H1CDR3)，所述第一轻链可变区包含第一轻链可变区的互补决定区1(L1CDR1)、第一轻链可变区的互补决定区2(L1CDR2)和/或第一轻链可变区的互补决定区3(L1CDR3)；和所述抗PD-L1抗体或其抗原结合片段为特异性结合PD-L1的抗体或其抗原结合片段。
在一些实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(a2)如SEQ ID NO:10、2和11所示的氨基酸序列；
(a3)如SEQ ID NO:4、5和6所示的氨基酸序列；和
(a4)如SEQ ID NO:7、8和9所示的氨基酸序列；
(a5)与(a1)、(a2)、(a3)或(a4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a6)如SEQ ID NO:12、13和14所示的氨基酸序列；
(a7)如SEQ ID NO:15、16和17所示的氨基酸序列；和
(a8)如SEQ ID NO:18、19和20所示的氨基酸序列；
(a9)与(a6)、(a7)或(a8)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段具有：
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:12、13和14或与SEQ ID NO:12、13和14所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:10、2和11或与SEQ ID NO:10、2和11所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:12、13和14或与SEQ ID NO:12、13和14所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；或
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:18、19和20或与SEQ ID NO:18、19和20所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的 第一轻链可变区。
在一些实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:27所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:28所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
所述第一重链可变区的氨基酸序列为SEQ ID NO:21，SEQ ID NO:21经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:21功能相同的氨基酸序列或与SEQ ID NO:21具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性的氨基酸序列；
所述第一重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性的氨基酸序列；
所述第一重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列；
所述第一重链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:24，SEQ ID NO:24经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:24功能相同的氨基酸序列或与SEQ ID NO:24具有至少85％序列同一性的氨基酸序列；或
所述第一重链可变区的氨基酸序列为SEQ ID NO:23，SEQ ID NO:23经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:23功能相同的氨基酸序列或与SEQ ID NO:23具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段为人源化抗体或其抗原结合片段，其包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得 的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段为人源化抗体或其抗原结合片段，其包含第一重链可变区和第一轻链可变区，其中
所述第一重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列；
所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性的氨基酸序列；或
所述第一重链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:34，SEQ ID NO:34经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:34功能相同的氨基酸序列或与SEQ ID NO:34具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中：
所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区包含H1CDR1、H1CDR2和H1CDR3，其氨基酸序列分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区包含L1CDR1、L1CDR2和L1CDR3，其氨基酸序列分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些优选的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段如以上实施方案中所限定；和所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和/或第二轻链可变区，其中所述第二重链可变区包含第二重链可变区的互补决定区1(H2CDR1)、第二重链可变区的互补决定区2(H2CDR2)和/或第二重链可变区的互补决定区3(H2CDR3)，所述第二轻链可变区包含第二轻链可变区的互补决定区1(L2CDR1)、第二轻链可变区的互补决定区2(L2CDR2)和/或第二轻链可变区的互补决定区3(L2CDR3)区。
进一步优选地，在一些实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段如以上实施方案中所限定，和所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(A1)如SEQ ID NO:75、76和77所示的氨基酸序列；
(A2)如SEQ ID NO:81、82和83所示的氨基酸序列；
(A3)如SEQ ID NO:87、88和89所示的氨基酸序列；和
(A4)如SEQ ID NO:93、94和95所示的氨基酸序列；
(A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(A6)如SEQ ID NO:78、79和80所示的氨基酸序列；
(A7)如SEQ ID NO:84、85和86所示的氨基酸序列；
(A8)如SEQ ID NO:90、91和92所示的氨基酸序列；
(A9)如SEQ ID NO:96、97和98所示的氨基酸序列；
(A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括 抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中：
所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区包含H1CDR1、H1CDR2和H1CDR3，其氨基酸序列分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；
(2)所述第一轻链可变区包含L1CDR1、L1CDR2和L1CDR3，其氨基酸序列分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(A1)如SEQ ID NO:75、76和77所示的氨基酸序列；
(A2)如SEQ ID NO:81、82和83所示的氨基酸序列；
(A3)如SEQ ID NO:87、88和89所示的氨基酸序列；和
(A4)如SEQ ID NO:93、94和95所示的氨基酸序列；
(A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(A6)如SEQ ID NO:78、79和80所示的氨基酸序列；
(A7)如SEQ ID NO:84、85和86所示的氨基酸序列；
(A8)如SEQ ID NO:90、91和92所示的氨基酸序列；
(A9)如SEQ ID NO:96、97和98所示的氨基酸序列；
(A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一个具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为 SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:75、76和77或与SEQ ID NO:75、76和77所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:78、79和80或与SEQ ID NO:78、79和80所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:81、82和83或与SEQ ID NO:81、82和83所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:84、85和86或与SEQ ID NO:84、85和86所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:87、88和89或与SEQ ID NO:87、88和89所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ  ID NO:90、91和92或与SEQ ID NO:90、91和92所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中所述抗CD47抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第一轻链可变区；和所述抗PD-L1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:93、94和95或与SEQ ID NO:93、94和95所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:96、97和98或与SEQ ID NO:96、97和98所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
在一些具体的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段以及抗PD-L1抗体或其抗原结合片段各自独立地为鼠源抗体、嵌合抗体、人源化抗体或完全人抗体。
在一些具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其中所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:22和SEQ ID NO:30所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:25和SEQ ID NO:33所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区的氨基酸序列选自：
(B1)如SEQ ID NO:99、100、101、102、110、111、112、113、114、119、120、121、122和123所示的氨基酸序列；
(B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区的氨基酸序列选自：
(B4)如SEQ ID NO:103、104、105、106、115、116、117、118、124、125、126所示的氨基酸序列；
(B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ  ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:15、16和17所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，其中所述抗CD47抗体或抗PD-L1抗体可以为鼠源抗体，其还含有鼠源的IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和鼠源的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，其中所述抗CD47鼠源抗体还含有鼠源的IgG1或IgG2或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在一些具体的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
(1)所述第一重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:30所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第一轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:33所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
(1)所述第二重链可变区的氨基酸序列选自：
(C1)如SEQ ID NO:110、111、112、113、114、119、120、121、122和123所示的氨基酸序列；
(C2)(C1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、 且与(C1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(C3)与(C1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述第二轻链可变区的氨基酸序列选自：
(C4)如SEQ ID NO:115、116、117、118、124、125、126所示的氨基酸序列；
(C5)(C4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(C4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(C6)与(C4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:110、111、112、113或114所示的氨基酸序列，SEQ ID NO:110、111、112、113或114经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:110、111、112、113或114功能相同的氨基酸序列或与SEQ ID NO:110、111、112、113或114具有至少85％序列同一性的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:115、116、117或118，SEQ ID NO:115、116、117或118经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:115、116、117或118功能相同的氨基酸序列或与SEQ ID NO:115、116、117或118具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸 获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:119、120、121、122或123所示的氨基酸序列，SEQ ID NO:119、120、121、122或123经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:119、120、121、122或123功能相同的氨基酸序列或与SEQ ID NO:119、120、121、122或123具有至少85％序列同一性的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:124、125或126，SEQ ID NO:124、125或126经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:124、125或126功能相同的氨基酸序列或与SEQ ID NO:124、125或126具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:15、16和17所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:110、111、112、113或114所示的氨基酸序列，SEQ ID NO:110、111、112、113或114经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:110、111、112、113或114功能相同的氨基酸序列或与SEQ ID NO:110、111、112、113或114具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:75、76和77所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:115、116、117或118，SEQ ID NO:115、116、117或118经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:115、116、117或118功能相同的氨基酸序列或与SEQ ID NO:115、116、117或118具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:78、79和80所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添 加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:15、16和17所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:110、111、112、113或114所示的氨基酸序列，SEQ ID NO:110、111、112、113或114经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:110、111、112、113或114功能相同的氨基酸序列或与SEQ ID NO:110、111、112、113或114具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:87、88和89所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:115、116、117或118，SEQ ID NO:115、116、117或118经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:115、116、117或118功能相同的氨基酸序列或与SEQ ID NO:115、116、117或118具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:90、91和92所示的氨基酸序列。
在一些优选的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:15、16和17所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:112所示的氨基酸序列，SEQ ID NO:112经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:112功能相同的氨基酸序列或与SEQ ID NO:112具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:75、 76和77所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:116，SEQ ID NO:116经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:116功能相同的氨基酸序列或与SEQ ID NO:116具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:78、79和80所示的氨基酸序列。
在另一些优选的实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述第一重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:4、5和6所示的氨基酸序列，且所述第一轻链可变区的氨基酸序列为SEQ ID NO:33，SEQ ID NO:33经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:33功能相同的氨基酸序列或与SEQ ID NO:33具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:15、16和17所示的氨基酸序列；和所述第二重链可变区的氨基酸序列为SEQ ID NO:123所示的氨基酸序列，SEQ ID NO:123经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:123功能相同的氨基酸序列或与SEQ ID NO:123具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:87、88和89所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:126，SEQ ID NO:126经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:126功能相同的氨基酸序列或与SEQ ID NO:126具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:90、91和92所示的氨基酸序列。
在一些实施方案中，本发明提供一种抗CD47/抗PD-L1人源化抗体，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的鼠源抗CD47/抗PD-L1抗体，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区。
在本发明一个优选的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗CD47抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其突变序列的重链恒定区，优选包含人源IgG1、IgG2、IgG4重链恒定区。
在一些具体的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗CD47人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗CD47人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗PD-L1抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2、IgG3、IgG4或其突变序列的重链恒定区，优选包含人源IgG或其突变序列的重链恒定区；所述抗PD-L1抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。
在一些具体的实施方案中，根据本发明的抗CD47/抗PD-L1抗体，所述抗PD-L1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗CD47人源化抗体或其抗原结合片段还包含人源IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗CD47/抗PD-L1抗体，其中所述的抗CD47抗体或其抗原结合片段以及抗PD-L1抗体或其抗原结合片段分别为Fab、Fv、sFv或F(ab) 2。在一个具体的实施方案中，本发明提供的抗CD47/抗PD-L1抗体为scF(ab) 2。
优选地，本发明以上实施方案中的抗CD47/抗PD-L1抗体是抗CD47/抗PD-L1双特异性抗体。在一些实施方案中，所述双特异性抗体是人抗体或人源化抗体。在一些实施方案中，所述结合特异性之一是针对CD47，而另一个结合特异性是针对任何其它抗原。在一些实施方案中，所述结合特异性之一是针对CD47，而另一个结合特异性是针对PD-L1。在一些实施方案中，所述双特异性抗体可结合 CD47的两种不同表位。所述双特异性抗体还可用于将细胞毒剂定位于表达CD47的细胞。这些抗体拥有CD47结合臂和细胞毒剂结合臂，所述细胞毒剂例如肥皂草毒蛋白、抗干扰素-α、长春花生物碱类、蓖麻毒蛋白A链、甲氨蝶呤或放射性同位素半抗原。可将本发明的双特异性抗体制备成全长抗体或抗体片段(例如F(ab') 2双特异性抗体)。
制备双特异性抗体的方法是本领域已知的。传统上，双特异性抗体的重组制备基于两个免疫球蛋白重链-轻链对的共表达，其中两个重链具有不同的特异性(Millstein and Cuello,Nature 305:537(1983))。由于免疫球蛋白重链和轻链的随机分配，这些杂交瘤(quadroma))可能产生10种不同抗体分子的混合物，其中只有一种分子具有正确的双特异性结构。该正确分子的纯化通常通过亲和层析步骤进行，相当麻烦且产物产量低。类似的方法在WO93/08829及Trauneckeretal.,EMBOJ.10:3655(1991)中有公开。
根据一种不同的方法，具有期望结合特异性(抗体-抗原结合位点)的抗体可变区与免疫球蛋白恒定区序列融合。在一些实施方案中，与包含至少部分铰链、CH2和CH3区的免疫球蛋白重链恒定区进行融合。在一些实施方案中，包含与轻链结合所必需位点的第一重链恒定区(CH1)存在于该融合的至少一部分中。将编码免疫球蛋白重链融合片段以及免疫球蛋白轻链(如果需要)的DNA***不同的表达载体中并共转染入合适的宿主生物体。在用于构建的三种多肽链比例不等时提供最佳产量的实施方案中，这为调整三种多肽片段的相互比例提供极大的灵活性。不过，在至少两种多肽链以相同比例表达产生高产量时或比例没有特别意义时，有可能将两种或所有三种多肽链的编码序列***一个表达载体中。
在该方法的一个实施方案中，所述双特异性抗体由一个臂中具有第一结合特异性的杂合免疫球蛋白重链和另一个臂中的杂合免疫球蛋白重链-轻链对(提供第二结合特异性)组成。由于免疫球蛋白轻链仅在该双特异性分子的一半中存在提供了便利的分离途径，因此发现该不对称性结构便于将期望的双特异性物质与不想要的免疫球蛋白链组合物分开。该方法在WO 94/04690中公开。关于产生双特异性抗体的进一步信息参见例如Sureshetal.,Methods in Enzymology 121:210(1986)。
根据另一种方法，可改造一对抗体分子间的界面以使从重组细胞培养物回收 的异二聚体的百分比最大化。该界面包含抗体恒定区CH3结构域的至少一部分。在该方法中，将第一抗体分子界面的一个或多个小氨基酸侧链用较大侧链(例如酪氨酸或色氨酸)替换。通过将大氨基酸侧链用较小氨基酸侧链(例如丙氨酸或苏氨酸)替换，在第二抗体分子的界面上产生与大侧链相同或相似大小的补偿性“空腔”。这提供了提高异二聚体相比于其它不想要的终产物诸如同二聚体的产量的机制。
双特异性抗体包括交联或“异源缀合”抗体。例如，一种异源缀合抗体可以与亲合素偶联，另一种异源缀合抗体可以与生物素偶联。可使用任何便利的交联方法来制备异源缀合抗体。合适的交联剂是本领域众所周知的，连同许多交联技术一起在美国专利No.4,676,980中公开。
本发明的双特异性抗体可以由抗体片段生成。例如，可使用化学连接技术来制备双特异性抗体。Brennanetal.,Science 229:81(1985)描述了通过蛋白水解切割完整抗体以生成F(ab') 2片段的方法。将这些片段在存在二硫醇络合剂***的情况下(用以稳定邻近的二硫醇和防止分子间二硫键的形成)分解。然后将产生的Fab'片段转变为硫代硝基苯甲酸酯(TNB)衍生物。然后将Fab'-TNB衍生物之一通过巯基乙胺的还原重新恢复成Fab'-硫醇，并与等摩尔量的另一种Fab'-TNB衍生物混合，以形成双特异性抗体。
可以从大肠杆菌直接回收Fab'-SH片段，这些片段可化学偶联以形成双特异性抗体。Shalaby et al.,J.Exp.Med.175:217-225(1992)描述了完全人源化的双特异性抗体F(ab') 2分子的生成。每个Fab'片段由大肠杆菌单独分泌，并在体外进行定向化学偶联以形成双特异性抗体。
在一些实施方案中，本发明的双特异性抗体片段可以直接从重组细胞培养物生成和分离。例如，可以使用亮氨酸拉链生成双特异性抗体(Kostelnyetal.,J.Immunol.148(5):1547-1553(1992))。将来自Fos和Jun蛋白的亮氨酸拉链肽通过基因融合与两种不同抗体的Fab'部分连接。抗体同二聚体在铰链区分解以形成单体，然后重新氧化以形成抗体异二聚体。该方法也可用于生成抗体同二聚体。双抗体技术提供了制备双特异性抗体片段的其他机制。所述双特异性抗体片段包含通过接头相连的重链可变区(VH)和轻链可变区(VL)，所述接头太短以使得同一条链上的两个结构域之间不能配对。因此，迫使一个片段上的VH和VL结构 域与另一个片段上的互补VL和VH结构域配对，由此形成两个抗原结合位点。在另一实施方案中，可以通过使用单链Fv(sFv)二聚体构建双特异性抗体片段。
本发明涵盖具有超过两价的多价抗体，例如，可制备三特异性抗体。多价抗体可以比二价抗体更快的受到表达该抗体结合的抗原的细胞的内在化(和/或异化)。本发明的抗体可以是可容易地通过编码抗体多肽链的核酸的重组表达而生成的、具有三个或更多抗原结合位点(例如四价抗体)的多价抗体。多价抗体可包含二聚化结构域和三个或更多抗原结合位点。在一些实施方案中，二聚化结构域包含(或由其组成)Fc区或铰链区。在这种情况中，抗体会包含Fc区及Fc区氨基末端的三个或更多抗原结合位点。在一些实施方案中，多价抗体包含(或由其组成)三个至大约八个抗原结合位点。在一些实施方案中，多价抗体包含四个抗原结合位点。多价抗体包含至少一条多肽链(例如两条多肽链)，其中所述多肽链包含两个或更多可变区。本发明的多价抗体可进一步包含至少两条(例如四条)轻链可变区多肽。本发明的多价抗体可包含例如约两条至约八条轻链可变区多肽。本发明的轻链可变区多肽包含轻链可变区，且任选进一步包含CL结构域。
在包含CD47靶向部分和PD-L1靶向部分的双特异性抗体中，CD47靶向部分和PD-L1靶向部分之一可以是全长抗体，并且另一个可以是包含重链CDR、轻链CDR或其组合的抗原结合片段(例如scFv)。靶向CD47和PD-L1蛋白之一的全长抗体和靶向另一蛋白的抗原结合片段可以直接或通过肽接头以化学方式连接(例如共价连接)。抗原结合片段(例如scFv)可以直接或通过肽接头与全长抗体的N-末端(例如全长抗体的轻链或重链的N-末端)、全长抗体的C-末端(例如全长抗体的重链(或Fc或CH3结构域)的C-末端)或两者连接。
在一个实施方案中，双特异性抗体可以包含全长抗CD47抗体、抗PD-L1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的肽接头。在其他实施方案中，双特异性抗体可以包含全长抗CD47抗体、抗PD-L1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的肽接头。
在一个实施方案中，双特异性抗体中包含的scFv可以按任何顺序包含重链可变区和轻链可变区。例如，双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含重链可变区和轻链可变区以及任选地在它们之间的肽接头，或者可替代地，双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包 含轻链可变区和重链可变区以及任选地在它们之间的肽接头。
在一些实施方案中，所述肽接头可包括例如Gly、Asn和/或Ser残基，并且还可以包括中性氨基酸，例如Thr和/或Ala。适用于肽接头的氨基酸序列可以是相关领域中已知的那些。同时，可以在使得融合蛋白功能不受影响的这样的限度内不同地确定肽接头的长度。例如，肽接头可以通过包括总共约1至约100、约2至约50、或约5至约25个选自由Gly、Asn、Ser、Thr、和Ala组成的组的一种或多种来形成。在一个实施方案中，肽接头可以表示为(GmSl)n(m、l和n独立地是约1至约10的整数，特别是约2至约5的整数)。
在另一个实施方案中，PD-L1靶向部分和CD47靶向部分可以均是全长抗体或包含重链CDR、轻链CDR或其组合的抗原结合片段。
在另一个实施方案中，双特异性抗体可以是异二聚体形式，其包含第一臂和第二臂，该第一臂包括靶向CD47和PD-L1之一的一对第一重链和第一轻链，该第二臂包括靶向另一者的一对第二重链和第二轻链。
在一个实施方案中，全长抗体可以是全长免疫球蛋白形式(例如IgG、IgM、IgA、IgE或IgD，例如人IgG、人IgM、人IgA、人IgE或人IgD)，并且抗原结合片段可以选自由Fab、Fab’、F(ab’) 2、Fd、Fv、scFv、scFab、单链抗体、sdFv等组成的组。例如，全长抗体可以是全长人IgG(人IgG1、人IgG2、人IgG3或人IgG4)形式，并且抗原结合片段可以是scFv。
例如，本文描述的抗体可以包含柔性接头序列，或者可以被修饰以添加功能部分(例如PEG、药物、毒素或标记)。
在一些具体的实施方案中，根据本发明的抗CD47/抗PD-L1双特异性抗体，所述抗CD47抗体或其抗原结合片段的结构为(VL-CL)-肽接头-(VH)-IgG4CH，所述抗PD-L1抗体或其抗原结合片段的结构为(VL-CL)-肽接头-(VH)-IgG4CH。在一些具体的实施方案中，所述肽接头为(GGGGS)n形式，其中n为1-12，优选为3-10，更优选为6-8，例如6、7、8个GGGGS重复序列。在另一些具体的实施方案中，靶向CD47部分的(VL-CL)-肽接头-(VH)-IgG4CH中的IgG4CH为含有S228P、L235E、Y349C、T366S、L368A、Y407V突变形成“Hole”结构的IgG4CH段，靶向PD-L1部分的(VL-CL)-肽接头-(VH)-IgG4CH中的IgG4CH为含有S228P、L235E、T366W、S354C突变形成“Knob”结构的IgG4CH段。在一些具体的实施方案中， 靶向CD47部分的(VL-CL)-肽接头-(VH)-IgG4CH中的VL的氨基酸序列为SEQ ID NO：33，CL的氨基酸序列为SEQ ID NO:131，VH的氨基酸序列为SEQ ID NO：30，IgG4CH的氨基酸序列为SEQ ID NO:133；和/或靶向PD-L1部分的(VL-CL)-肽接头-(VH)-IgG4CH中的VL的氨基酸序列为SEQ ID NO：116，CL的氨基酸序列为SEQ ID NO:131，VH的氨基酸序列为SEQ ID NO：112，IgG4CH的氨基酸序列为SEQ ID NO:132。在另一些具体的实施方案中，靶向CD47部分的(VL-CL)-肽接头-(VH)-IgG4CH中的VL的氨基酸序列为SEQ ID NO：33，CL的氨基酸序列为SEQ ID NO:131，VH的氨基酸序列为SEQ ID NO：30，IgG4CH的氨基酸序列为SEQ ID NO:133；和/或靶向PD-L1部分的(VL-CL)-肽接头-(VH)-IgG4CH中的VL的氨基酸序列为SEQ ID NO：126，CL的氨基酸序列为SEQ ID NO:131，VH的氨基酸序列为SEQ ID NO：123，IgG4CH的氨基酸序列为SEQ ID NO:132。
本发明的另一方面提供抗PD-L1抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的CDR1、CDR2和CDR3：
(A1)如SEQ ID NO:75、76和77所示的氨基酸序列；
(A2)如SEQ ID NO:81、82和83所示的氨基酸序列；
(A3)如SEQ ID NO:87、88和89所示的氨基酸序列；和
(A4)如SEQ ID NO:93、94和95所示的氨基酸序列；
(A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的CDR1、CDR2和CDR3：
(A6)如SEQ ID NO:78、79和80所示的氨基酸序列；
(A7)如SEQ ID NO:84、85和86所示的氨基酸序列；
(A8)如SEQ ID NO:90、91和92所示的氨基酸序列；
(A9)如SEQ ID NO:96、97和98所示的氨基酸序列；
(A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中所述抗PD-L1抗体或其抗原结合片段包含：
所述CDR1、CDR2和CDR3分别为SEQ ID NO:75、76和77或与SEQ ID NO:75、 76和77所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述CDR1、CDR2和CDR3分别为SEQ ID NO:78、79和80或与SEQ ID NO:78、79和80所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；
所述CDR1、CDR2和CDR3分别为SEQ ID NO:87、88和89或与SEQ ID NO:87、88和89所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述CDR1、CDR2和CDR3分别为SEQ ID NO:90、91和92或与SEQ ID NO:90、91和92所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；或者
所述CDR1、CDR2和CDR3分别为SEQ ID NO:93、94和95或与SEQ ID NO:93、94和95所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述CDR1、CDR2和CDR3分别为SEQ ID NO:96、97和98或与SEQ ID NO:96、97和98所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中：
(1)所述重链可变区的氨基酸序列选自：
(B1)如SEQ ID NO:99、100、101、102、110、111、112、113、114、119、120、121、122和123所示的氨基酸序列；
(B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(B4)如SEQ ID NO:103、104、105、106、115、116、117、118、124、125、126所示的氨基酸序列；
(B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中：
所述重链可变区的氨基酸序列为SEQ ID NO:110、111、112、113或114所示的氨基酸序列，SEQ ID NO:110、111、112、113或114经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:110、111、112、113或114功能相同 的氨基酸序列或与SEQ ID NO:110、111、112、113或114具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:115、116、117或118，SEQ ID NO:115、116、117或118经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:115、116、117或118功能相同的氨基酸序列或与SEQ ID NO:115、116、117或118具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中：
所述重链可变区的氨基酸序列为SEQ ID NO:119、120、121、122或123所示的氨基酸序列，SEQ ID NO:119、120、121、122或123经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:119、120、121、122或123功能相同的氨基酸序列或与SEQ ID NO:119、120、121、122或123具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:124、125或126，SEQ ID NO:124、125或126经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:124、125或126功能相同的氨基酸序列或与SEQ ID NO:124、125或126具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:112所示的氨基酸序列，SEQ ID NO:112经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:112功能相同的氨基酸序列或与SEQ ID NO:112具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:75、76和77所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:116，SEQ ID NO:116经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:116功能相同的氨基酸序列或与SEQ ID NO:116具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:78、79和80所示的氨基酸序列。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:123所示的氨基酸序列，SEQ ID NO:123经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:123功能相同的氨基酸序列或与SEQ ID NO:123具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:87、88和89所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:126，SEQ ID  NO:126经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:126功能相同的氨基酸序列或与SEQ ID NO:126具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:90、91和92所示的氨基酸序列。
在一些实施方案中，根据本发明的抗PD-L1抗体或其抗原结合片段，其中所述抗体是人源化抗体或完全人抗体。
本发明的另一方面提供分离的核酸。在一些实施方案中，根据本发明的分离的核酸编码本发明的抗CD47/抗PD-L1抗体或抗PD-L1抗体。在一些实施方案中，根据本发明的分离的核酸编码本发明的抗CD47抗体或其抗原结合片段。在另一些实施方案中，根据本发明的分离的核酸编码本发明的抗PD-L1抗体或其抗原结合片段。
在一个具体的实施方案中，根据本发明的分离的核酸，编码第一重链可变区SEQ ID NO:30的核苷酸序列如SEQ ID NO:36所示，且编码第一轻链可变区SEQ ID NO:33的核苷酸序列如SEQ ID NO:39所示。在另一个具体的实施方案中，根据本发明的分离的核酸，编码第二重链可变区SEQ ID NO:112的核苷酸序列如SEQ ID NO:127所示，且编码第二轻链可变区SEQ ID NO:116的核苷酸序列如SEQ ID NO:129所示。在再一个具体的实施方案中，根据本发明的分离的核酸，编码第二重链可变区SEQ ID NO:123的核苷酸序列如SEQ ID NO:128所示，且编码第二轻链可变区SEQ ID NO:126的核苷酸序列如SEQ ID NO:130所示。
本发明的另一方面提供表达载体。在一些实施方案中，本发明的表达载体表达本发明的抗CD47/抗PD-L1双特异性抗体或抗PD-L1抗体。在一些实施方案中，本发明的表达载体表达本发明的抗CD47抗体或其抗原结合片段。在另一些实施方案中，本发明的表达载体表达本发明的抗PD-L1抗体或其抗原结合片段。在一些实施方案中，根据本发明的表达载体，表达本发明的抗CD47抗体或其抗原结合片段的载体和表达本发明的抗PD-L1抗体或其抗原结合片段的载体是同种表达载体。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗CD47/抗PD-L1双特异性抗体或抗PD-L1抗体的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗CD47/抗PD-L1双特异性抗体和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗CD47/抗PD-L1双特异性抗体，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
本发明的另一方面提供一种嵌合抗原受体(CAR)融合蛋白，其包含本发明的抗CD47抗体或其抗原结合片段和/或抗PD-L1抗体或其抗原结合片段。在一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗CD47抗体或其抗原结合片段，其为针对CD47抗原的V H和V L的单链可变片段(scFv)。在另一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗PD-L1抗体或其抗原结合片段，其为针对PD-L1抗原的V H和V L的单链可变片段(scFv)。在另一些实施方案中，所述嵌合抗原受体融合蛋白包含针对CD47抗原的V H和V L的第一单链可变片段(scFv)和针对PD-L1抗原的V H和V L的第二单链可变片段(scFv)。所述针对CD47抗原的V H和V L的第一scFv具有以上实施方案中描述的第一重链可变区的H1CDR1、H1CDR2和H1CDR3和第一轻链可变区的L1CDR1、L1CDR2和L1CDR3。所述针对PD-L1抗原的V H和V L的第二scFv具有以上实施方案中描述的第二重链可变区的H2CDR1、H2CDR2和H2CDR3和第二轻链可变区的L2CDR1、L2CDR2和L2CDR3。
可以将本发明的抗CD47/抗PD-L1双特异性抗体与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供治疗和/或预防与CD47、PD-L1或两者相关的疾病的方法，所述方法包括向有此需要的个体施用本发明的抗CD47/抗PD-L1双特异性抗体或本发明的药物组合物。
本发明的另一方面提供本发明的抗CD47/抗PD-L1双特异性抗体或抗PD-L1抗体或本发明的药物组合物在制备治疗和/或预防与CD47、PD-L1或两者相关的疾病的药物中的应用。在一些实施方案中，所述与CD47、PD-L1或两者相关的疾病包括血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。所述肿瘤可以是任何表达PD-L1蛋白的肿瘤，例如膀胱癌、肝癌、结肠癌、直肠癌、子宫内膜癌、白血病、淋巴瘤、胰腺癌、肺癌(例如小细胞肺癌、非小细胞肺癌等)、乳腺癌、尿道癌、头颈癌、胃肠癌、胃癌、食道癌、卵巢癌、肾癌、黑素瘤、***癌、甲状腺癌等。所述肿瘤可以是原发性或转移性肿瘤。在一些实施方案中，本发明提供上述抗CD47/抗PD-L1双特异性抗体或本发明的药物组合物在制备抗肿瘤的药物中的应用，例如所述肿瘤选自血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌、神经胶质瘤和黑素瘤。
本发明提供的抗CD47/抗PD-L1双特异性抗体具有显著的抗肿瘤作用，可明显抑制肿瘤生长，且不具有明显的红细胞毒性，人源化后的抗体免疫原性大大降低，有效消除人体免疫***对外源性单抗的排异反应，可在制备用于治疗各类肿瘤疾病的药物中应用，具有广阔的市场前景。
定义
除非另有定义，本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual)(第2版，Cold Spring Harbor Laboratory Press，纽约冷泉港(1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中 使用的命名以及实验室方法和技术是本领域公知且普遍使用的。
在本发明中，术语“至少80％序列同一性”是指至少80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在本发明中，术语“至少85％序列同一性”是指至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在一些优选的实施方案中，本发明所述的序列同一性可以至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。
在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补，且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院，马里兰州贝塞斯达(1987和1991))或Chothia和Lesk，J.Mol.Biol.196:901-917(1987)，Chothia等，Nature 342:878-883(1989)定义。
本发明的“抗体”是指特异性地识别并结合抗原的多肽或多肽复合物。抗体可以是完整抗体和其任何抗原结合片段或单链。本发明的“抗体”包括含有Ig分子的具有结合抗原的生物活性的至少一部分的任何蛋白质或肽。本发明“抗体”的实例包括但不限于重链或轻链的CDR或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分。
本发明所述的“抗原结合片段”包括具有抗原结合活性的Fab片段、Fab’片段、F(ab’)2片段及与人CD47或PD-L1结合的Fv片段、scFv片段。Fv片段含有抗体第一重链可变区和第一轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体或单链Fv(scFv)。本发明的抗CD47或抗PD-L1抗体可以是单链可变区片段(scFv)，其源自抗体的单链多肽，保留了结合抗原的能力。scFv的实例包括通过重组DNA技术形成的抗体多肽，其中免疫球蛋白重链(H链)和轻链(L链)片段的Fv区经由间隔序列连接。制备scFv的各种 方法是本领域技术人员所熟知的。
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10 -6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。
本发明所述的“鼠源抗体”为根据本领域知识和技能制备的对人CD47的单克隆抗体。制备时用CD47抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。
本发明所述的“人源化抗体”也称为CDR移植抗体，是将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。此类可变区框架序列可以从公共的DNA数据库或公开的参考文献获得，例如从ImMunoGeneTics(IMGT)网站http://imgt.cines.fr得到或从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明所述的“双特异性抗体”指对至少两种不同抗原具有结合特异性的单克隆抗体。
本发明所述的“肽接头”可以是包括1至10，特别是2至50个任何氨基酸的那些，并且可以包括任何种类的氨基酸而没有任何限制。
附图说明
图1是抗CD47人源化抗体与猴CD47结合活性测定(ELISA)结果。
图2是抗CD47人源化抗体与人CD47结合活性测定(ELISA)结果。
图3是抗CD47人源化抗体与细胞表面CD47结合活性测定(ELISA)结果。
图4是红细胞凝集实验结果，其中RBC：阳性对照；PBS：空白对照。
图5是FACS检测抗CD47人源化抗体阻断活性实验结果。
图6是抗CD47人源化抗体Hu34-39-PE人胃癌NUGC-4移植瘤模型抗肿瘤试验结果。
图7是抗CD47人源化抗体Hu26T-31-PE人胃癌NUGC-4移植瘤模型抗肿瘤试验结果。
图8是双抗ScFab(HuPL7-21Ks/Hu34-39Hs)的结构示意图。
图9是ScFabHuPL7-21Ks序列示意图。
图10是ScFabHu34-39Hs序列示意图。
图11是双抗ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)与PD-L1结合(A)、与PD-1竞争(B)、与CD47结合(C)、与SIRPα竞争(D)以及与CD80竞争(E)实验结果。
图12是在细胞水平双抗ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)与细胞表面PD-L1结合(A)、与细胞表面CD47结合(B)以及阻断PD-1与PD-L1结合(C)、阻断SIRPα与CD47结合(D)的实验结果。
图13是双抗ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)在Raji-hPD-L1细胞上的CD47/PD-L1双结合(A、B)及CD47/PD-L1双阻断(B)实验结果。
图14是双抗ScFab(HuPL7-21Ks/Hu34-39Hs)对小鼠体内移植瘤生长抑制实验结果。
具体实施方式
下面代表性的实施例是为了更好地说明本发明，而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件，如冷泉港的抗体技术实验手册、分子克隆手册等，或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。
实施例1 CD47抗原蛋白及抗CD47阳性对照抗体的制备
1、抗原蛋白及阳性对照抗体的表达载体构建
(1)抗原蛋白的表达载体构建
合成编码CD47蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:41所示，然后克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-CD47。
融合人源CD47蛋白胞外区氨基酸序列与hIgG1-Fc或his标签氨基酸序列，氨基酸序列设计分别如SEQ ID NO:42和SEQ ID NO:43所示。对上述氨基酸序列进行密码子优化后合成带有标签的CD47蛋白胞外区基因片段CD47-hFc、CD47-his，并将其分别克隆至真核表达质粒pHR中，获得其表达质粒pHR-CD47-hFc、pHR-CD47-his。
融合人源CD47蛋白胞外区氨基酸序列与mIgG1-Fc氨基酸序列，氨基酸序列设计如SEQ ID NO:44所示。对该氨基酸序列进行密码子优化后合成完整的表达质粒pcDNA3.1(+)-TPA-CD47-mIgG1-Fc。
SIRPα的序列如SEQ ID NO:45所示，对所述序列进行密码子优化后合成完整的表达质粒pcDNA3.1(+)-SIRPα-myc-His。
(2)阳性对照抗体的表达载体构建
使用专利申请WO2013/119714中公开的抗体AB6.12-IgG4P(本文简称AB06.12-4P)作为阳性对照抗体，AB06.12-4P的氨基酸序列如下所示：
AB06.12-4P重链氨基酸序列：SEQ ID NO:46；
AB06.12-4P轻链氨基酸序列：SEQ ID NO:47。
对上述抗体序列所对应的氨基酸序列进行密码子人工优化，获得阳性对照抗体AB06.12-4P的重链及轻链表达质粒pcDNA3.1(+)-SHC025-hG4、pcDNA3.1(+)-SHC025-hk。并将其重链基因片段克隆到含有IgG4轻链恒定区的真核表达质粒pHR上，获得AB06.12-4P的重链真核表达质粒pHR-SHC025-hG4-4PE，其轻链表达质粒为pcDNA3.1(+)-SHC025-hk。
2、抗原蛋白及阳性对照抗体的表达与纯化
(1)抗原蛋白的稳转细胞株的构建
将真核表达质粒pTargeT-CD47在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置37℃，5％ CO 2的培养箱中培养。24h后采用含500ug/ml G418的培养基加压培养。16天后采用FACS检测pool阳性率，将电转质粒后的细胞铺板(1x10 6个/ml的细胞密度，100ul/孔)，采用PE mouse anti-human CD47抗体(BD，556046)与细胞孵育，以流式细胞仪(BD，FACSJazz)读取585nm波长下mean值，使用GraphPad进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达CD47分子，命名为CHO-K1-E5。
(2)标签抗原蛋白及阳性对照抗体的表达
在1L细胞培养瓶中接种密度为0.5 x 10 6个细胞/ml的293F细胞，加入新鲜的预热的FreeStyle 293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8.5mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500ul，混合均匀，取250ug待转染质粒加入8.5ml FreeStyle 293表达培养基中，混合均匀，其中标签抗原蛋白质粒pHR-CD47-hFc、pHR-CD47-his、pcDNA3.1(+)-TPA-CD47-mIgG1-Fc、pcDNA3.1(+)-SIRPα-myc-His分别转染；阳性对照抗体AB06.12-4P重链质粒pHR-SHC025-hG4-4PE和轻链质粒pcDNA3.1(+)-SHC025-hk共同转染。将PEI与FreeStyle 293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)和5ml的葡萄糖(母液浓度为180g/L)。当细胞细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
(3)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用不同的亲和层析柱进行纯化(不同蛋白适配的亲和层析柱见表1)，具体纯化步骤如下：
表1 不同蛋白适配的亲和层析柱
Figure PCTCN2021078132-appb-000001
Figure PCTCN2021078132-appb-000002
清洗：超纯水清洗设备及管路2min，流速10mL/min，后用0.1M NaOH清洗层析***；
接柱：将层析柱接入层析设备，并用超纯水冲洗5min；后0.1M NaOH冲洗30min，保留时间5min；
平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV(柱体积)；
上样：将细胞表达上清上样，保留时间5min；
后平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV；
洗脱：50mM醋酸，pH＝3.4洗脱，保留时间5min。UV280至50mAu左右时开始收集，降至50mAu左右时停止收集。用1M Tris-HCl，pH 9.0将样品pH调节至7.0；
再平衡：20mM PB+0.15M NaCl，pH 7.2平衡3个CV，保留时间5min；
在线清洗：0.1M NaOH清洗30min，保留时间5min；
清洗保存：纯化水清洗10min，后20％乙醇2个CV。
实施例2 抗CD47单克隆抗体的制备
1、杂交瘤单克隆的制备
(1)动物免疫
采用不同标签的抗CD47抗原蛋白与佐剂共同免疫的方法免疫SJL品系的实验小鼠，首次抗原使用50ug抗原，后期使用25ug抗原免疫。
免疫佐剂可以是Quick Antibody-Mouse5W(北京博奥龙免疫技术有限公司)或Titer Max(Sigma)与CpG(金斯瑞生物科技有限合成)/Alum(thermo)佐剂间隔。将不同标签的CD47抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫SJL小鼠。
高水平表达CD47分子的细胞系如CCRF-CEM和CHO-K1-E5也用来免疫小鼠，使之产生抗体。用胰蛋白酶消化处理正在培养的人急性淋巴白血病细胞(CCRF-CEM)和实施例1中获得的CHO-K1-E5阳性单细胞，1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，取样用细胞计数仪计数，剩余样品1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，计入适量的PBS以获得1x10 8个细胞/ml 的细胞悬液。实验组小鼠每只免疫1x10 7个细胞。
免疫方案如表2所示：
表2 小鼠免疫方案
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。
组别 抗原 佐剂 免疫途径*
1 PBS 无  
2 CD47-his/CD47-mFc Quick Antibody-Mouse5W i.m.
3 CD47-his/CD47-mFc Titer Max/CpG/Alum s.c./i.m.
4 CD47-mFc Quick Antibody-Mouse5W i.m.
5 CD47-mFc Titer Max/CpG/Alum s.c./i.m.
6 CCRF-CEM/CHO-K1-E5 无 i.p.
(2)杂交瘤融合
脾细胞的获取和制备：将加强免疫后的小鼠处死后浸泡75％的酒精中。解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1:1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL RPMI1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
(3)杂交瘤及亚克隆上清的筛选
融合一周后，取细胞上清，通过ELISA筛选出能结合CD47-his蛋白或细胞表面CD47的杂交瘤上清，利用CD47-his筛选针对CD47而非hFc、mFc的抗体。然后利用ELISA分析杂交瘤上清阻断CD47-SIRPα相互作用的能力。包被SIRP α-myc-his于酶标板上，加入重组人源蛋白CD47-hFc与杂交瘤上清的混合物孵育2h，加入HRP标记的anti human IgG Fc特异性抗体(Jackson Immuno Research)孵育1h，利用酶标仪检测450nm处的吸光值。将筛选获得的具有结合能力及阻断能力的杂交瘤母克隆扩大培养，进行结合活性及阻断活性的复测，再次筛选获得具有结合及阻断能力的杂交瘤阳性克隆。
利用有限稀释法将阳性细胞株进行亚克隆，培养一周后利用ELISA检测亚克隆上清与CD47分子的结合活性以及阻断CD47-SIRPα相互作用的活性，获得3种双阳性细胞株，分别标记为SHC025-26、SHC025-34、SHC025-58。
2、亚型的鉴定
参照鼠抗体亚型鉴定试剂盒SBA Clonotyping Systerm-C57BL/6-HRP(SouthernBiotech，货号：5300-05B)说明书对抗体进行亚型鉴定，结果如表3所示：
表3 抗体亚型鉴定结果
命名 抗体亚型
SHC025-26 IgG1/k
SHC025-34 IgG2c/k
SHC025-58 IgG2b/k
3、单克隆抗体的制备
根据亚克隆上清活性分析结果确定单克隆抗体母克隆株SHC025-26、SHC025-34、SHC025-58，将其扩大培养。培养条件是含有10％胎牛血清、1x NAEE、1x丙酮酸钠、1％青链霉素双抗的1640培养基，待细胞汇合度大于>80％时，进将细胞传代扩培，待培养至约50ml时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。
4、单克隆抗体测序
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据小鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体 发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得小鼠抗体轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester etal.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.Simon KorenMiha
Figure PCTCN2021078132-appb-000003
Colja Venturini etal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列，测序结果见表4。
表4 抗CD47鼠源单克隆抗体序列表
抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHC025-26 SEQ ID NO:21 SEQ ID NO:24
SHC025-34 SEQ ID NO:22 SEQ ID NO:25
SHC025-58 SEQ ID NO:23 SEQ ID NO:26
抗体SHC025-34的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:4、5、6，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:15、16、17。实施例3 抗CD47嵌合抗体的构建
将纯化后(纯化步骤见实施例1)的小鼠抗体轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将混合液均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序；将测序正确的嵌合抗体分别标记为SHC025-26CHI、SHC025-34CHI、SHC025-58CHI。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得嵌合抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将嵌合抗体转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
测序发现，在SHC025-26的重链CDR第118位，以及SHC025-58的轻链CDR第56位各自含有一个半胱氨酸，CDR区域的半胱氨酸在表达时会与抗体分子上 的其他半胱氨酸随机配对氧化形成二硫桥，从而极大的影响抗体纯度。为解决之一问题，对SHC025-26CHI、SHC025-58CHI氨基酸序列进行如下改造：将SHC025-26CHI重链之C118突变为T，标记为SHC025-26CHI-T；将SHC025-58CHI轻链之C56突变为A，标记为SHC025-58CHI-A，利用定点突变的方法构建突变基因。嵌合抗体测序结果见表5。
表5 抗CD47嵌合抗体序列表
嵌合抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHC025-26CHI SEQ ID NO:21 SEQ ID NO:24
SHC025-26CHI-T SEQ ID NO:27 SEQ ID NO:24
SHC025-34CHI SEQ ID NO:22 SEQ ID NO:25
SHC025-58CHI SEQ ID NO:23 SEQ ID NO:26
SHC025-58CHI-A SEQ ID NO:23 SEQ ID NO:28
抗体SHC025-34CHI的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:4、5、6，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:15、16、17。
实施例4 抗CD47人源化抗体的构建及生产
根据嵌合抗体的活性分析、亲和力KD值等结果选择SHC025-34CHI、SHC025-58CHI-A、SHC025-26CHI-T进行人源化抗体改造。
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对，确认SHC025-34CHI、SHC025-58CHI-A、SHC025-26CHI-T抗体可变区的鼠源种系，经过同源比对，SHC025-34CHI、SHC025-58CHI-A、SHC025-26CHI-T抗体的重链可变区序列的FR区分别与人抗体胚系基因IGHV1-8*01、IGHV3-21*04以及IGHV1-2*02最为相似；抗体轻链可变区的FR序列则分别与人抗体胚系基因IGKV3-11*01、IGKV1-5*01以及IGKV4-1*01最为相似。以SHC025-34CHI/SHC025-58CHI-A抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代SHC025-34CHI/SHC025-58CHI-A的FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，最终选择SHC025-34CHI的6条人源化重链可变区(参见SEQ ID NO:48、49、 50、51、52、53)和4条人源化轻链可变区(参见SEQ ID NO:54、55、56、57)及SHC025-58CHI-A的6条人源化重链可变区(参见SEQ ID NO:58、59、60、61、62、63)和5条人源化轻链可变区(参见SEQ ID NO:64、65、66、67、68)进行下一步优化。SHC025-34CHI/SHC025-58CHI-A人源化抗体非CDR区序列均达到95％以上人源化。用SHC025-26CHI-T重轻链的可变区序列，在Protein Data Bank中进行结构比对分析，选择最相近的FR1-FR3序列替换掉鼠源序列，并将在结构模拟中显示对抗体结构稳定起到关键作用的的氨基酸位点回突变为鼠源性氨基酸残基。最终获得SHC025-26CHI-T的4条人源化重链可变区(参见SEQ ID NO:69、70、71、72)和2条人源化轻链可变区(参见SEQ ID NO:73、74)。
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有IgG4轻链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与含S228P/L235E突变的IgG4重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
挑选纯度、活性和亲和力均较好的人源化抗体，标记为Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE，序列见表6。人源化抗体来源见表7。
表6 抗CD47人源化抗体序列表
Figure PCTCN2021078132-appb-000004
Figure PCTCN2021078132-appb-000005
抗体Hu34-39-PE的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:4、5、6，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:15、16、17。
表7 人源化序列设计信息
Figure PCTCN2021078132-appb-000006
实施例5 抗CD47抗体与猴CD47结合活性测定(ELISA)
采用protein based Elisa分析抗体的结合活性。食蟹猴CD47-His(0.1μg/孔，ACRO Biosystems，Cat.No.CD7-C52H1-50ug)包被96孔酶标板。本发明提供的抗CD47抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h，阳性对照抗体为AB06.12-4P。二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB(3,3',5,5'-四甲基联苯胺)，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50，结果如图1所示。
实验结果显示本发明提供的人源化抗CD47抗体Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均具有与食蟹猴CD47结合的能力，且结合能力与阳性对照抗体AB06.12-4P相当。
实施例6 抗CD47抗体与人CD47结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人CD47-His蛋白(0.1ug/孔，实施例1、2中制得)包被到96孔酶标板，37℃孵育2h。用1xPBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1xPBST清洗3次后，本发明提供的抗CD47抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h，阳性对照抗体为AB06.12-4P；用1xPBST清洗5次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，37℃孵育40min。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50，结果如图2所示。
实验结果显示本发明提供的人源化抗CD47抗体Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均具有与人CD47结合的能力，且结合能力与阳性对照抗体AB06.12-4P相当。
实施例7 抗CD47抗体与细胞表面CD47结合的测定(ELISA)
采用Cell based Elisa分析抗体的结合活性。CHO-K1-E5细胞以每孔1x10 5个细胞的方式铺细胞板，置于37℃，5％CO 2的条件下过夜培养；第二天用4％的多聚甲醛固定后用脱脂牛奶封闭1h；用1xPBS轻柔清洗细胞板；本发明提供的抗CD47抗体作为一抗从2μg/mL开始，5倍梯度稀释加入细胞板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0ng/mL，37℃孵育1.5h，阳性对照抗体为AB06.12-4P；二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50，结果如图3所示。
实验结果显示，本发明提供的人源化抗CD47抗体Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均可与细胞表面CD47结合，且结合能力与阳性对照抗体AB06.12-4P相当。
实施例8 抗CD47抗体与人CD47蛋白的亲和力测定
利用Fortebio Octet对实施例1、2中制得的人源化抗CD47抗体结合抗原CD47(19-136)-hFC的亲和力进行测定。先将抗原CD47(19-136)-hFc生物素化标 记，然后用10kD的超滤管，PBS离心超滤脱盐3-4次，Nanodrop测定生物素标记后抗原CD47-hFc-Biotin的实际浓度。将CD47-hFc-Biotin用SD缓冲液(0.02％Tween20+0.1％BSA溶液)稀释到浓度5ug/ml，所述人源化抗CD47抗体用SD缓冲液4倍浓度梯度稀释，使其浓度为10ug/ml、2.5ug/ml、0.625ug/ml、0ug/ml，选用SA传感器固化该抗原，按fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表8所示。
表8 与人CD47蛋白的亲和力测定
抗体 KD(M) kon(1/Ms) kdis(1/s)
Hu26T-31-PE 5.87E-11** 1.04E+06 6.13E-05
Hu34-39-PE 1.13E-10 6.69E+06 7.58E-04
Hu58A-14-PE 9.46E-11 1.78E+06 1.68E-04
AB06.12-4P 1.01E-10 4.80E+06 4.85E-04
实验结果显示，与阳性对照抗体相比，人源化抗CD47抗体Hu26T-31-PE与人CD47蛋白结合具有更高的亲和力。
实施例9 抗CD47抗体的红细胞凝集实验
取5mL血液加入40mL PBS，2000rpm轻柔离心5min，弃上清，用PBS洗涤三次，然后用PBS重悬红细胞，按红细胞积压，配制2％的红细胞悬液。待分析抗体起始浓度为1-20uM之间，2倍梯度稀释，共24个浓度梯度。圆底96孔板中加入上述50μL不同浓度抗体，然后加入50ul上述2％的红细胞悬液，混匀，室温放置。2h后观察是否有凝集现象。兔多克隆抗体RBC抗体(Rockland，109-4139)作为红细胞凝集发生的阳性对照，结果如图4所示。如图4所示，96孔板从左至右加入的抗体(兔多克隆抗体RBC抗体、AB06.12-4P抗体及本发明待测抗体)浓度从20uM起始2倍梯度依次稀释，其中RBC表示阳性对照组(使用兔多克隆抗体RBC抗体，显著引起红细胞凝集)，PBS表示空白对照组。孔中红细胞小圆点状，边缘整齐表示不引起细胞凝集；边缘略不整齐表示少量红细胞凝集；片状，布满孔底表示大部分红细胞凝集。
经测定，专利申请WO2011/143624中公开的抗CD47抗体Hu5F9-G4在相同浓度范围内能引起显著的大部分红细胞凝集，此为抗CD47抗体的普遍不良现象。 而在相同的条件下，如实验结果显示，本发明的Hu26T-31-PE、Hu34-39-PE、Hu58A-14-PE均不会引起红细胞凝集现象，本发明的抗体在此方面明显优于抗体Hu5F9-G4。
实施例10 抗CD47抗体对CD47阻断活性的测定
采用FACS检测本发明提供的抗CD47抗体阻断SIRPa结合到细胞表面CD47的能力。
CHO-K1-E5阳性细胞株作为CD47提供者，在梯度稀释的抗CD47抗体存在的情况下，观察CD47与SIRPa的结合能力。二抗使用PE Streptavidin(Biolegend，405203，1:200)来监测SIRPa-Biotin的变化。AB06.12-4P作为阻断SIRPa结合到细胞表面CD47的阳性对照。流式细胞仪(BD，FACSJazz)读取585nm波长下mean值，使用GraphPad生成IC50，结果如图5所示。
实验结果显示，阻断活性依次排序为Hu26T-31-PE≧Hu34-39-PE≧AB06.12-P﹥Hu58A-14-PE。
实施例11 抗CD47抗体在人胃癌NUGC-4移植瘤模型中抗肿瘤试验
1、实验材料
(1)实验细胞及动物
NUGC-4人胃癌细胞购自美国典型培养物保藏中心(ATCC)；
NOD-Scid小鼠，雌性，5-8周龄，体重18-20克，购自上海灵畅生物科技有限公司；
(2)供试品及对照品
对照品Isotype IgG4(货号AB170091)购自中美冠科生物技术有限公司，用作阴性对照品；
试验前，将本发明的人源化抗CD47抗体用PBS配制为0.6mg/mL和0.3mg/mL两个浓度，Isotype IgG4和AB 06.12-4P配制为0.6mg/mL。
(3)实验方法
用含有10％胎牛血清，100U/mL的青霉素和100μg/mL的链霉素的RMPI1640培养基在37℃、5％CO 2的培养箱中培养NUGC-4人胃癌细胞。一周一次用2mL 1×EDTA溶液消化处理传代。当细胞饱和度为80％-90％时，收取细胞，计数，接种。将含有5×10 6细胞PBS同100uL的Matrigel混合(终体积200μL)接种于小 鼠的右后边，接种细胞数目为5×10 6/只。待肿瘤生长至体积达150-200mm 3时开始分组，腹腔给药每周3次，每周三次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。抗体的抑瘤疗效用相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。RTV＝V21/V0，其中V0是分组给药时(即d0)测量所得肿瘤体积，V21为给药21天测量时的肿瘤体积。将给药组与溶媒组最后一天(day21)瘤体积用T-test进行分析，用GraphPad Prism进行。结果如表9所示。
表9 人胃癌NUGC-4移植瘤模型抗肿瘤试验结果
Figure PCTCN2021078132-appb-000007
***p＜0.001vs.Isotype IgG4；**p＜0.005vs.Isotype IgG4
实验结果显示，本发明提供的抗体在人胃癌NUGC-4细胞接种NOD-SCID小鼠移植瘤模型中具有较显著的抗肿瘤作用。3mg/kg剂量的Hu26T-31-PE和Hu34-39-PE与6mg/kg的参照抗体AB06.12-4P抑瘤作用相当，而6mg/kg剂量Hu26T-31-PE和Hu34-39-PE抑瘤作用优于6mg/kg参照抗体AB06.12-4P。停药一周后，6mg/kg的参照抗体组出现肿瘤复发现象，而Hu26T-31-PE和Hu34-39-PE组无复发现象(图6、图7)。提示本发明提供的抗CD47抗体出乎意料的具有更加显著抑制肿瘤生长的作用。
实施例12 抗人PD-L1抗体的获得
1.动物免疫
采用mFc标签的PD-L1抗原蛋白(购于北京百普赛斯生物科技有限公司)与佐剂共同免疫的方法免疫BALB/c、SJL品系的实验小鼠和SD品系的实验大鼠。
免疫佐剂首次是Freund’s Adjuvant,Complete(SIGMA,F5881-10ML)，后期用Freund’s Adjuvant,Incomplete(SIGMA,F5506-10ML)。将不同标签的PD-L1抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫小鼠或大鼠。免疫方案如表10所示：
表10 免疫方案
Figure PCTCN2021078132-appb-000008
*i.m.肌内注射
2.细胞融合
无菌取小鼠脾脏并制成细胞悬液，按脾细胞：Sp2/0细胞＝1:1的比例进行细胞融合。将融合后的细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
3.阳性克隆筛选
融合一周后，取细胞上清，通过ELISA筛选出具有结合及阻断能力的杂交瘤 母克隆细胞并进行扩大培养，复测结合活性及阻断活性，再次筛选获得具有结合及阻断能力的杂交瘤阳性细胞株。利用有限稀释法将阳性细胞株进行亚克隆，培养一周后用ELISA检测亚克隆上清与PD-L1分子的结合活性以及阻断PD-L1/PD-1相互作用的活性，获得PL-7、PL-15、PL-16、PL-18四株优选双阳性细胞株。
4.抗PD-L1抗体可变区序列的获得
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据小鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得小鼠抗体轻链与重链可变区的基因片段，命名为SHS009PL-7、SHS009PL-15、SHS009PL-16、SHS009PL-18；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester etal.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.Simon KorenMiha
Figure PCTCN2021078132-appb-000009
Colja Venturini etal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列，测序结果见表11。
表11 抗PD-L1鼠源单克隆抗体序列表
Figure PCTCN2021078132-appb-000010
Figure PCTCN2021078132-appb-000011
5.嵌合抗体的构建
将纯化后的小鼠抗体轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区基因片段的真核表达质粒共转化大肠杆菌DH5α感受态细胞，挑取测序正确的嵌合抗体分别标记为PL-7CHI、PL-15CHI、PL-16CHI、PL-18CHI，嵌合抗体测序结果见表12。
PL-7CHI、PL-15CHI、PL-16CHI、PL-18CHI轻重链可变区氨基酸序列，分别同鼠源性抗体SHS009PL-7、SHS009PL-15、SHS009PL-16、SHS009PL-18轻重链可变区氨基酸序列一致。
提取嵌合抗体轻链质粒与重链质粒转染HEK 293F细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
表12 抗PD-L1嵌合抗体序列表
嵌合抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
PL-7CHI SEQ ID NO：99 SEQ ID NO：103
PL-15CHI SEQ ID NO：100 SEQ ID NO：104
PL-16CHI SEQ ID NO：101 SEQ ID NO：105
PL-18CHI SEQ ID NO：102 SEQ ID NO：106
6.抗PD-L1抗体人源化改造
根据嵌合抗体的活性分析、亲和力KD值等结果选择PL-7CHI、PL-16CHI进行人源化抗体改造。
鼠源单克隆嵌合抗体PL-7CHI、PL-16CHI的人源化参照经典的CDR移植策略进行，以PL-7CHI、PL-16CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代PL-7CHI/PL-16CHI的FR1-FR3序列。经同源比对分析，PL-7CHI、PL-16CHI抗体重链可变区FR区分别与人抗体种系基因M99683|IGHV4-31*02(SEQ ID NO:107)、X62109|IGHV1-3*01(SEQ ID NO:108)最为相似，PL-7CHI、PL-16CHI抗体轻链可变区FR区与人 抗体种系基因Z00023|IGKV4-1*01(SEQ ID NO:109)最为相似。人源化后的重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，并将在结构模拟中显示对抗体结构稳定起到关键作用的的氨基酸位点回突变为鼠源性氨基酸残基。最后得到PL-7CHI人源化重链5条(PL-7CHI人源化重链可变区序列：VH1-0(SEQ ID NO:110)、VH1-1(SEQ ID NO:111)、VH1-2(SEQ ID NO:112)、VH1-3(SEQ ID NO:113)或VH1-4(SEQ ID NO:114))、人源化轻链4条(PL-7CHI人源化轻链可变区序列：VL1-0(SEQ ID NO:115)、VL1-1(SEQ ID NO:116)、VL1-2(SEQ ID NO:117)或VL1-3(SEQ ID NO:118))；PL-16CHI人源化重链5条(PL-16CHI人源化重链可变区序列：VH1-0(SEQ ID NO:119)、VH1-1(SEQ ID NO:120)、VH1-2(SEQ ID NO:121)、VH1-3(SEQ ID NO:122)或VH1-4(SEQ ID NO:123))、人源化轻链3条(PL-16CHI人源化轻链可变区序列：VL1-0(SEQ ID NO:124)、VL1-1(SEQ ID NO:125)或VL1-2(SEQ ID NO:126))。在此基础上，通过不同的轻重链组合，我们得到了多个人源化抗体。经活性检测后，确定其中评分最高的抗PD-L1人源化抗体序列分别为HuPL7-21、HuPL16-42。
实施例13 人源化抗PD-L1抗体生产
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列合成相对应的核苷酸编码序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有IgG4轻链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与含S228P/L235E突变的IgG4重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293F细胞，表达纯化获得大量抗体，进行纯度检测、活性 分析及亲和力的检测。序列见表13。
表13 抗PD-L1人源化抗体序列表
Figure PCTCN2021078132-appb-000012
抗体HuPL7-21的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:75、76和77，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:78、79和80。抗体HuPL16-42的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:87、88和89，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:90、91和92。
实施例14 抗CD47/PD-L1双特异性抗体的构建、表达及纯化
1.双特异性抗CD47/抗PD-L1抗体表达载体的构建
运用基因工程手段构建抗CD47/抗PD-L1的双特异性抗体，其结构形式如图8所示：该双特异性抗体是由两条分别抗PD-L1和抗CD47的抗体单链通过异源二聚化而形成；区别于天然IgG抗体，该双特异性抗体中抗PD-L1和抗CD47抗体的轻链都通过额外添加的柔性连接肽连接至抗体重链N端，该连接肽是含甘氨酸(G)和丝氨酸(S)残基的GGGGS重复序列，优选包含8个GGGGS的重复序列；此外，为促进异源二聚体的形成，在上述S228P/L235E突变的基础上，在抗PD-L1抗体单链的CH3domain再增加S354C/T366W突变，在抗CD47抗体单链的CH3domain再增加Y349C/T366S/L368A/Y407V突变。根据该结构形式，利用上述抗PD-L1抗体HuPL7-21(轻链可变区SEQ ID NO：116和恒定区CL SEQ ID NO:131)或HuPL16-42(轻链可变区SEQ ID NO：126和恒定区CL SEQ ID NO:131)序列，通过基因合成获得抗PD-L1的抗体单链的核苷酸片段，即ScFabHuPL7-21Ks或ScFabHuPL16-42Ks；利用将上述抗CD47抗体Hu34-39(轻链可变区SEQ ID NO：33和和恒定区CL SEQ ID NO:131)序列，通过基因合成获得抗CD47的抗体单链的核苷酸片段，即ScFabHu34-39Hs。将该ScFabHuPL7-21Ks(或ScFabHuPL16-42Ks)、ScFabHu34-39Hs核苷酸片段(上下游均含有适当长度的同源臂)分别与线性化的真核表达质粒pHR共转化大肠杆菌DH5α感受态细胞， 分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。将测序正确的阳性克隆进行质粒提取，从而获得ScFabHuPL7-21Ks(或ScFabHuPL16-42Ks)、ScFabHu34-39Hs表达载体。
ScFabHuPL7-21Ks序列示意图如图9所示，
其中：HuPL7-21VL-CL：人源化PD-L1单抗HuPL7-21轻链；
(GGGGS)8：8个GGGGS重复序列的柔性连接肽；
HuPL7-21VH：人源化PD-L1单抗HuPL7-21重链可变区；
IgG4CH/Ks：含有S228P/L235E/S354C/T366W突变形成“Knobs”结构的IgG4重链恒定区(具体序列例如SEQ ID NO:132)；
ScFabHu34-39Hs序列示意图如图10所示，
其中：Hu34-39VL-CL：人源化CD47单抗Hu34-39轻链；
(GGGGS)6：6个GGGGS重复序列的柔性连接肽；
Hu34-39VH：人源化CD47单抗Hu34-39重链可变区；
IgG4CH/Hs：含有S228P/L235E/Y349C/T366S/L368A/Y407V突变形成
“Hole”结构的IgG4重链恒定区(具体序列如SEQ ID NO:133)。
以上结构中的CL序列如SEQ ID NO:131所示。
2.抗CD47/抗PD-L1双特异性抗体在Expi-CHO细胞中的瞬时表达
利用Expi-Fectamine CHO Transfection Kit质粒转染试剂盒将上述的ScFabHuPL7-21Ks(或ScFabHuPL16-42Ks)和ScFabHu34-39Hs两个表达载体共重组质粒转染Expi-CHO细胞，在无血清培养基中培养14天后收集Expi-CHO细胞上清液，免疫印迹检测双特异性抗体的表达情况。其中，将由ScFabHuPL7-21Ks和ScFabHu34-39Hs两个单链二聚化而形成的双特异性抗体命名为ScFab(HuPL7-21Ks/Hu34-39Hs)，将由ScFabHuPL16-42Ks和ScFabHu34-39Hs两个单链二聚化而形成的双特异性抗体命名为ScFab(HuPL16-42Ks/Hu34-39Hs)。
3.双特异性抗CD47/抗PD-L1抗体的纯化
本发明的双特异性抗体在Expi-CHO细胞中表达并分泌后，采用Protein A亲和层析的方法对其进行纯化，具体方法如下：Protein A亲和层析柱用缓冲 液平衡后，将超滤器浓缩过的Expi-CHO细胞培养液上清液进行进样，并以A280(nm)进行监测，用清洗液洗至未结合的蛋白全部被洗脱，然后用洗脱液进行洗脱，以获得相应的双特异性抗体。纯化后的双特异性抗体SEC-HPLC检测纯度，LC-MS检测分子量，进行质量鉴定后用于后续的药学研究。SEC-HPLC、LC-MS鉴定结果表明，双抗ScFab(HuPL7-21Ks/Hu34-39Hs)和ScFab(HuPL16-42Ks/Hu34-39Hs)的纯度均达到95％以上，分子量测定值与理论值匹配。
实施例15 高表达hPD-L1稳转细胞株的构建
1.高表达hPD-L1稳转细胞株CHO-K1-hPD-L1、Raji-hPD-L1的构建：
将含hPD-L1(人PD-L1)胞外区序列(UniProtKB-Q9NZQ7(PD-L1 Human)>sp|Q9NZQ7|19-238，SEQ ID NO:134)的真核表达质粒pTargeT-hPD-L1以电转的方式转染到CHO-K1细胞(来自中国科学院上海细胞生物学研究所)及Raji细胞(来自宜明昂科生物医药技术有限公司)中，置37℃，5％CO 2的培养箱中培养。24h后采用含500ug/ml G418的培养基加压培养。12天后采用FACS检测pool阳性率，将电转质粒后的细胞铺板(1x10 6个/ml的细胞密度，100ul/孔)，将FITC anti-human PD-L1抗体(SINO BIOLOGICAL，10084-MMB6-F)与细胞在4℃孵育60min，以流式细胞仪读取FITC通道下mean值，对结果进行数据分析后，挑出阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1/Raji细胞株，该细胞株高水平表达PD-L1分子，命名为CHO-K1-hPD-L1和Raji-hPD-L1。
实施例16 抗CD47/抗PD-L1双特异性抗体ELISA体外结合与阻断实验
1.抗CD47/抗PD-L1双特异性抗体PD-L1结合ELISA实验
将人PD-L1-His蛋白(0.5ug/ml，100ul/孔)包被到96孔酶标板，37℃孵育2h。用1xPBST清洗3次后用5％的脱脂牛奶4℃封闭过夜，用1xPBST清洗3次。双抗浓度从10μg/mL开始，5倍梯度稀释加入酶标板，37℃孵1.5h，对照抗体为Atezolizumab(Sino Biological，Cat:68049-H001，简写Ate)；用1xPBST清洗5次后，加入HRP-Anti-Human IgG二抗(Jackson，109-035-003，1:10000)，37℃孵育40min。用1xPBST清洗5次，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值，EC 50结果如图11所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、 ScFab(HuPL16-42Ks/Hu34-39Hs)均具有与人PD-L1结合的能力。与人PD-L1结合能力与Atezolizumab相当。
2.抗CD47/抗PD-L1双特异性抗体与PD-1竞争ELISA实验
采用ELISA分析抗体阻断PD1与PD-L1结合的活性。将人PD-L1-hFC蛋白(2ug/ml，100ul/孔)包被到96孔酶标板，37℃孵育2h。用1xPBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1xPBST清洗3次后，用双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)或ScFab(HuPL16-42Ks/Hu34-39Hs)作为一抗从10μg/mL开始，3倍梯度稀释，共8个浓度，在梯度稀释的抗PD-L1抗体存在的情况下，与1μg/mL的PD-1-mFc共同加入酶标板37℃孵1.5h，对照抗体为Atezolizumab；用1xPBST清洗5次后，二抗使用Anti-Mouse IgG HRP(Jackson，109-035-003，1:10000)，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪读取OD450值。结果如图11所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)均具有阻断PD-L1与PD-1结合的能力，且阻断能力与Atezolizumab相当。
3.抗CD47/抗PD-L1双特异性抗体CD47结合ELISA实验
采用ELISA分析抗体与CD47结合活性。将人CD47-His蛋白(0.5ug/ml，100ul/孔)包被到96孔酶标板，37℃孵育2h。用1xPBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1xPBST清洗3次后，本发明提供的抗CD47/抗PD-L1双抗浓度从50μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，37℃孵育1.5h，对照抗体为Hu34-39-PE；用1xPBST清洗5次后，二抗使用HRP-Anti-Human IgG(1:10000)，37℃孵育40min。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。EC 50结果如图11所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)均具有与人CD47结合的能力，结合活性均有不同程度的降低，ScFab(HuPL7-21Ks/Hu34-39Hs)结合EC 50值较Hu34-39-PE的EC 50值高40倍。
4.抗CD47/抗PD-L1双特异性抗体与SIRPα竞争ELISA实验
采用ELISA分析抗体的阻断活性。将人CD47-His蛋白(0.4ug/ml，100ul/孔)包被到96孔酶标板，37℃孵育2h。用1xPBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1xPBST清洗3次后，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)或ScFab(HuPL16-42Ks/Hu34-39Hs)作为一抗从10μg/mL开始，3倍梯度稀释，共8个浓度，在梯度稀释的抗CD47抗体存在的情况下，与2μg/mL的SIRPα-biotin共同加入酶标板37℃孵1.5h，对照抗体为Hu34-39-PE；用1xPBST清洗5次后，二抗使用SA-HRP(Jackson，109-035-003，1:10000)，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪读取OD450值，结果如图11所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)均具有阻断CD47与SIRPα结合的能力，且阻断能力较Hu34-39-PE有所下降，IC 50值升高约40倍。
5.抗CD47/抗PD-L1双特异性抗体与CD80竞争ELISA实验
采用ELISA分析抗体阻断PD-L1与CD80结合的活性。将CD80-hFc蛋白(8ug/ml，100ul/孔)包被到96孔酶标板，4℃过夜孵育。用1xPBST清洗3次后用5％的脱脂牛奶37℃封闭2h。用1xPBST清洗3次后，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)或ScFab(HuPL16-42Ks/Hu34-39Hs)作为一抗从30μg/mL开始，3倍梯度稀释，共8个浓度，在梯度稀释的抗PD-L1抗体存在的情况下，与PD-L1-mFc共同加入酶标板37℃孵1.5h，对照抗体为Atezolizumab；用1xPBST清洗5次后，二抗使用Anti-Mouse IgG HRP，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪读取OD450值。结果如图11所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)均具有阻断PD-L1与CD80结合的能力，且阻断能力与Atezolizumab相当。
以上实验结果表明，本发明的双抗可以差异性结合PD-L1/CD47，从而在保证抗肿瘤活性的同时，可能降低抗体的毒性反应，如血液方面的毒性等。
实施例17 抗CD47/抗PD-L1双特异性抗体细胞水平的结合/阻断实验
1.采用FACS检测双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、 ScFab(HuPL16-42Ks/Hu34-39Hs)结合细胞表面PD-L1/CD47的能力。
CHO-K1-hPD-L1/CHO-K1-hCD47稳转细胞株作为PD-L1/CD47提供者，将梯度稀释的抗PD-L1/抗CD47双抗作为一抗加入细胞板，4℃孵育1.5h。二抗使用PE Anti-Human IgG，4℃孵育1h。Atezolizumab、Hu34-39-PE作为阳性对照。流式细胞仪读取585nm波长下mean值和Parent值的乘积，结果如图12所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)均具有与细胞表面的人PD-L1结合能力，且结合能力与Atezolizumab相当。双抗ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)与细胞表面CD47结合活性较Hu34-39-PE单抗结合活性降低，EC 50升高约4倍，Emax降低约2倍。
2.采用FACS检测双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)阻断PD-1/SIRPα结合到细胞表面的能力。
CHO-K1-hPD-L1/CHO-K1-hCD47稳转细胞株作为PD-L1/CD47提供者，在梯度稀释的抗PD-L1抗体/抗CD47抗体存在的情况下，观察PD-L1与PD-1结合、CD47与SIRPα结合的能力情况。双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)或ScFab(HuPL16-42Ks/Hu34-39Hs)作为一抗，梯度稀释后，分别与1μg/mL的PD-1-mFc、SIRPα-biotin共同加入细胞板中37℃孵1.5h，二抗使用PE-Anti-Mouse IgG/PE-SA。Atezolizumab作为阻断PD-1-mFc结合到细胞表面PD-L1的阳性对照，Hu34-39-PE作为阻断SIRPα结合到细胞表面CD47的阳性参照，结果如图12所示。
实验结果显示，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)均具有阻断人PD-1与CHO-K1-PD-L1、SIRPα与CHO-K1-CD47结合的能力，且阻断PD-1与CHO-K1-PD-L1结合的能力与Atezolizumab相当。双抗阻断SIRPα与CHO-K1-CD47结合的能力较Hu34-39-PE有所降低，IC 50值增加3倍。
根据ELISA及FACS检测的结果，双抗分子ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)与PD-L1结合与阻断能力与Atezolizumab一致。而双抗分子同CD47的结合能力有所下降。ELISA检测EC 50升高约40倍，FACS检测EC 50升高约4倍，Emax降低2倍。即双抗分子ScFab(HuPL7-21Ks/Hu34-39Hs)、 ScFab(HuPL16-42Ks/Hu34-39Hs)差异性结合PD-L1/CD47，这有助于增强双抗的肿瘤靶向性和降低双抗的不良反应，特别是针对红细胞的不良反应。
实施例18 FACS检测抗CD47/抗PD-L1双特异性抗体与Raji-hPD-L1细胞双结合及双阻断实验
Raji-hPD-L1肿瘤细胞购自宜明昂科生物医药技术有限公司，Raji-hPD-L1细胞表面除了hPD-L1高表达之外，hCD47也同样高表达，双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)、ScFab(HuPL16-42Ks/Hu34-39Hs)与Raji-hPD-L1细胞表面的hPD-L1和hCD47都结合，而且也能同时阻断CD47/SIRPα的结合和PD-1/PD-L1的结合，表现出双臂结合及双臂阻断的活性。双臂阻断：U型板底每孔铺2.4×10 5个Raji-hPD-L1细胞，加入一抗，抗体工作浓度从2.5μg/mL开始，2倍梯度稀释，共9个梯度，抗原终浓度1μg/mL的SIRPα-mFc与终浓度1μg/mL的PD-1-mFc均匀混合，抗体50μl与抗原50μl每孔预混，共100μl加入细胞孔中，于4℃孵育1.5h，加入二抗，200μl cell stain buffer洗涤三遍后，加每孔0.8μl PE-anti-mouse-IgG-Fc，于4℃孵育1h，200μl cell stain buffer洗涤三次，100μl cell stain buffer重悬，使用流式细胞仪检测；双臂结合：U型板底每孔铺1.5x10 6个Raji-hPD-L1细胞，加入一抗，抗体工作浓度从10μg/mL开始，5倍梯度稀释，共8个梯度，于4℃孵育1.5h，加入二抗，200μl cell stain buffer洗涤三遍后，加每孔0.8μl PE-anti-human-IgG-Fc，于4℃孵育1h，200μl cell stain buffer洗涤三次，100μl cell stain buffer重悬，使用流式细胞仪检测。
实验结果如图13所示。表明，本发明的双特异性抗体与双靶点细胞均结合，且能同时阻断双靶点细胞表面CD47/SIRPα结合及PD-1/PD-L1结合。
实施例19 抗CD47/抗PD-L1双特异性抗体对小鼠体内移植瘤生长抑制的检测
NSG小鼠具有NOD、Prkdcscid、IL2rgnull缺失/变异特征，是目前免疫缺陷程度最高、最适合人源细胞移植的工具小鼠，对人源细胞和组织几乎没有排斥反应。本发明利用NSG小鼠(购自北京百奥赛图基因生物技术有限公司)、Raji-hPD-L1肿瘤细胞建立肿瘤移植模型——Raji-PBMC-NSG模型，研究抗CD47/抗PD-L1双抗分子ScFab(HuPL7-21Ks/Hu34-39Hs)在Raji-hPD-L1淋巴瘤皮下移植模型中的抗肿瘤作用。CD47单抗5F9购自Sino Biological Inc.(Cat： 68063-H001)。表14为测试药物在Raji-PBMC-NSG肿瘤模型中的抗肿瘤作用实验设计方案。
表14 Raji-PBMC-NSG模型测试药物实验设计方案
Figure PCTCN2021078132-appb-000013
a：N是指每组小鼠的数量；b：BiAb指双抗ScFab(HuPL7-21Ks/Hu34-39Hs)
各组的肿瘤体积随时间的变化情况如图14所示。本发明的双抗体内抑瘤效果显著优于Atezolizumab，优于单抗联用(Atezolizumab+5F9)。
实施例20 抗CD47/抗PD-L1双特异性抗体食蟹猴急性毒性实验
本实施例为本发明的抗CD47/抗PD-L1双特异性抗体ScFab(HuPL7-21Ks/Hu34-39Hs)(简称BiAb)单次静脉输注给予食蟹猴的毒性试验。双抗的给药剂量为10、30、100mg/kg，食蟹猴每组雌雄各1只。抗CD47单抗Hu34-39-PE的给药剂量为30mg/kg，Hu5F9的给药剂量为20mg/kg，每组2只食蟹猴。
单次静脉输注，观察周期为21天。不同时间点经股静脉采血用于血细胞计数、凝血功能指标、血液生化指标等的检测。
药物安全性评价结果显示，截止第21天，所有组无猴死亡，一般状态观察、摄食量、体重等各组均无异常。
给予双抗ScFab(HuPL7-21Ks/Hu34-39Hs)的动物，RBC计数、HGB含量、RET％无变化。双抗ScFab(HuPL7-21Ks/Hu34-39Hs)在10、30、100mg/kg给药剂量下，未显示红细胞毒性及其它血液性毒性。
给予单抗Hu5F9(20mg/kg)的动物，RBC计数和HGB含量明显下降，RET％明显增加。给予单抗Hu34-39-PE(30mg/kg)的动物，RBC计数和HGB含量有一定程度的下降，RET％也有增加，但弱于单抗Hu5F9。Hu5F9显示出明显的红细胞 毒性，Hu34-39-PE红细胞毒性弱于Hu5F9。双抗ScFab(HuPL7-21Ks/Hu34-39Hs)的红细胞安全性显著优于单抗Hu34-39-PE和Hu5F9。
上述实施例表明，本发明的抗CD47/抗PD-L1双特异性抗体分子ScFab(HuPL7-21Ks/Hu34-39Hs)差异性结合CD47和PD-L1，较好地保留了与PD-L1的结合和阻断活性，同时没有红细胞毒性及其它血液性毒性，安全性优异。
实施例21 抗PD-L1抗体对小鼠体内移植瘤生长抑制的检测
本发明利用NSG小鼠(购自北京百奥赛图基因生物技术有限公司，中国)、Raji-PD-L1肿瘤细胞(购自宜明昂科生物医药技术有限公司，中国)建立肿瘤移植模型——Raji-PBMC-NSG模型，研究本发明的抗体在Raji-hPD-L1淋巴瘤皮下移植模型中的抗肿瘤作用。抗PD-L1阳性对照抗体为Atezolizumab(Sino Biological，Cat:68049-H001)。每组6只小鼠。阴性对照组给予生理盐水(PBS)。本发明的抗PD-L1抗体HuPL7-21和Atezolizumab的给药剂量分别为10mg/kg。给药途径腹腔注射，给药频次为每周2次，连续给药3周。
实验结束，计算各组的肿瘤抑制率(TGI TV)％，如表15所示。
表15 各抗体对Raji-PBMC-NSG模型小鼠肿瘤体积的影响
组别 剂量(mg/kg) TGI TV(％)
PBS - 0.00
Atezolizumab 10 37.40
HuPL7-21 10 48.75
本发明的抗PD-L1抗体HuPL7-21体内抑瘤效果显著优于Atezolizumab。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。

Claims (17)

  1. 一种抗CD47/抗PD-L1抗体，其包括抗CD47抗体或其抗原结合片段和抗PD-L1抗体或其抗原结合片段，其中：
    所述抗CD47抗体或其抗原结合片段包含第一重链可变区和第一轻链可变区，其中：
    (1)所述第一重链可变区包含H1CDR1、H1CDR2和H1CDR3，其氨基酸序列分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；
    (2)所述第一轻链可变区包含L1CDR1、L1CDR2和L1CDR3，其氨基酸序列分别为SEQ ID NO:15、16和17或与SEQ ID NO:15、16和17所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
  2. 如权利要求1所述的抗CD47/抗PD-L1抗体，其中所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
    (1)所述第二重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
    (A1)如SEQ ID NO:75、76和77所示的氨基酸序列；
    (A2)如SEQ ID NO:81、82和83所示的氨基酸序列；
    (A3)如SEQ ID NO:87、88和89所示的氨基酸序列；和
    (A4)如SEQ ID NO:93、94和95所示的氨基酸序列；
    (A5)与(A1)、(A2)、(A3)或(A4)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述第二轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
    (A6)如SEQ ID NO:78、79和80所示的氨基酸序列；
    (A7)如SEQ ID NO:84、85和86所示的氨基酸序列；
    (A8)如SEQ ID NO:90、91和92所示的氨基酸序列；
    (A9)如SEQ ID NO:96、97和98所示的氨基酸序列；
    (A10)与(A6)、(A7)、(A8)或(A9)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
  3. 如权利要求2所述的抗CD47/抗PD-L1抗体，其中所述抗PD-L1抗体或其抗原结合片段包含：
    所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:75、76和77或与SEQ ID NO:75、76和77所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:78、79和80或与SEQ ID NO:78、79和80所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区；
    所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:87、88和89或与SEQ ID NO:87、88和89所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:90、91和92或与SEQ ID NO:90、91和92所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区；
    所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:81、82和83或与SEQ ID NO:81、82和83所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:84、85和86或与SEQ ID NO:84、85和86所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区；或者
    所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:93、94和95或与SEQ ID NO:93、94和95所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:96、97和98或与SEQ ID NO:96、97和98所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的第二轻链可变区。
  4. 如权利要求1-3之任一项所述的抗CD47/抗PD-L1抗体，其中：
    (1)所述第一重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO:22和SEQ ID NO:30所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述第一轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO:25和SEQ ID NO:33所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  5. 如权利要求1-4之任一项所述的抗CD47/抗PD-L1抗体，其包括 抗PD-L1抗体或其抗原结合片段，其中：
    所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
    (1)所述第二重链可变区的氨基酸序列选自：
    (B1)如SEQ ID NO:99、100、101、102、110、111、112、113、114、119、120、121、122和123所示的氨基酸序列；
    (B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述第二轻链可变区的氨基酸序列选自：
    (B4)如SEQ ID NO:103、104、105、106、115、116、117、118、124、125、126所示的氨基酸序列；
    (B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  6. 如权利要求1-5之任一项所述的抗CD47/抗PD-L1抗体，其中：
    (1)所述第一重链可变区的氨基酸序列选自：
    (c1)如SEQ ID NO:30所示的氨基酸序列；
    (c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述第一轻链可变区的氨基酸序列选自：
    (c4)如SEQ ID NO:33所示的氨基酸序列；
    (c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  7. 如权利要求1-6之任一项所述的抗CD47/抗PD-L1抗体，其包括抗PD-L1抗体或其抗原结合片段，其中：
    所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
    所述第二重链可变区的氨基酸序列为SEQ ID NO:110、111、112、113或114所示的氨基酸序列，SEQ ID NO:110、111、112、113或114经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:110、111、112、113或114功能相同的氨基酸序列或与SEQ ID NO:110、111、112、113或114具有至少85％序列同一性的氨基酸序列且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:75、76和77所示 的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:115、116、117或118，SEQ ID NO:115、116、117或118经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:115、116、117或118功能相同的氨基酸序列或与SEQ ID NO:115、116、117或118具有至少85％序列同一性的氨基酸序列且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:78、79和80所示的氨基酸序列；或者
    所述第二重链可变区的氨基酸序列为SEQ ID NO:119、120、121、122或123所示的氨基酸序列，SEQ ID NO:119、120、121、122或123经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:119、120、121、122或123功能相同的氨基酸序列或与SEQ ID NO:119、120、121、122或123具有至少85％序列同一性的氨基酸序列且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:87、88和89所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:124、125或126，SEQ ID NO:124、125或126经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:124、125或126功能相同的氨基酸序列或与SEQ ID NO:124、125或126具有至少85％序列同一性的氨基酸序列且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:90、91和92所示的氨基酸序列。
  8. 如权利要求1-6之任一项所述的抗CD47/抗PD-L1抗体，其包括抗PD-L1抗体或其抗原结合片段，其中：
    所述抗PD-L1抗体或其抗原结合片段包含第二重链可变区和第二轻链可变区，其中：
    所述第二重链可变区的氨基酸序列为SEQ ID NO:112所示的氨基 酸序列，SEQ ID NO:112经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:112功能相同的氨基酸序列或与SEQ ID NO:112具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:75、76和77所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:116，SEQ ID NO:116经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:116功能相同的氨基酸序列或与SEQ ID NO:116具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:78、79和80所示的氨基酸序列；
    所述第二重链可变区的氨基酸序列为SEQ ID NO:123所示的氨基酸序列，SEQ ID NO:123经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:123功能相同的氨基酸序列或与SEQ ID NO:123具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:87、88和89所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:126，SEQ ID NO:126经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:126功能相同的氨基酸序列或与SEQ ID NO:126具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:90、91和92所示的氨基酸序列；或者
    所述第二重链可变区的氨基酸序列为SEQ ID NO:102所示的氨基酸序列，SEQ ID NO:102经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:102功能相同的氨基酸序列或与SEQ ID NO:102具 有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:93、94和95所示的氨基酸序列，且所述第二轻链可变区的氨基酸序列为SEQ ID NO:106，SEQ ID NO:106经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:106功能相同的氨基酸序列或与SEQ ID NO:106具有至少85％，或至少90％，或至少95％，或至少98％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:96、97和98所示的氨基酸序列。
  9. 如权利要求1-8之任一项所述的抗CD47/抗PD-L1抗体，其中所述抗体是人源化抗体或完全人抗体。
  10. 如权利要求1-9之任一项所述的抗CD47/抗PD-L1抗体，其中所述抗体是双特异性抗体。
  11. 一种分离的核酸，其编码权利要求1-10之任一项所述的抗CD47/抗PD-L1抗体。
  12. 如权利要求10所述的核酸，其中：
    (1)编码所述第一重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:36所示；
    (2)编码所述第一轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:39所示；
    (3)编码所述第二重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:127或SEQ ID NO:128所示；
    (4)编码所述第二轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:129或SEQ ID NO:130所示。
  13. 一种表达载体，其包含如权利要求11或12所述的核酸。
  14. 一种宿主细胞，其转化如权利要求13所述的表达载体，所述宿 主细胞选自原核细胞和真核细胞，优先为哺乳动物细胞。
  15. 制备权利要求1-10任一项所述的抗CD47/抗PD-L1抗体的方法，包括在如权利要求14所述的宿主细胞中表达抗体，以及从宿主细胞中分离所述抗体的步骤。
  16. 一种药物组合物，其包含权利要求1-10之任一项所述的抗CD47/抗PD-L1抗体和药学可接受的载体。
  17. 如权利要求1-10之任一项所述的抗CD47/抗PD-L1抗体或如权利要求16的药物组合物在制备用于抑制CD47和/或PD-L1活性的药物中的应用，优选所述药物用于治疗血液肿瘤、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。
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SHALABY ET AL., J. EXP. MED., vol. 175, 1992, pages 217 - 225
SIMON KORENMIHAKOSMACANJACOLJA VENTURINI ET AL., ANTIBODY VARIABLE-REGION SEQUENCING AS A METHOD FOR HYBRIDOMA CELL-LINE AUTHENTICATION, vol. 78, 2008, pages 1071 - 1078
SURESH ET AL., METHODS IN ENZYMOLOGY, vol. 121, 1986, pages 210
TRAUNECKER ET AL., EMBOJ, vol. 10, 1991, pages 3655

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EP4112647A4 (en) 2024-03-27

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