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WO2022100694A1 - 抗体及其制备方法 - Google Patents

抗体及其制备方法 Download PDF

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Publication number
WO2022100694A1
WO2022100694A1 PCT/CN2021/130320 CN2021130320W WO2022100694A1 WO 2022100694 A1 WO2022100694 A1 WO 2022100694A1 CN 2021130320 W CN2021130320 W CN 2021130320W WO 2022100694 A1 WO2022100694 A1 WO 2022100694A1
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antibody
human
variable region
heavy chain
light chain
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PCT/CN2021/130320
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English (en)
French (fr)
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王荣娟
王双
张畅
焦莎莎
曾大地
张姣
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迈威(上海)生物科技股份有限公司
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Priority to US18/036,576 priority Critical patent/US20230406921A1/en
Priority to EP21891214.5A priority patent/EP4253415A4/en
Publication of WO2022100694A1 publication Critical patent/WO2022100694A1/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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—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
    • C07K2317/56—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • 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 belongs to the field of antibody engineering, in particular to antibodies and preparation methods thereof, and more particularly, to humanized anti-human CD47 monoclonal antibodies or fragments thereof, multispecific antibodies, and bispecific antibodies targeting PD-L1 and CD47 or antigen-binding fragments thereof, polynucleotides, vectors, host cells, methods and compositions for preparing antibodies or antigen-binding fragments thereof, and related applications.
  • CD47 is highly expressed in most tumor cells, and it can be used as a standard for tumor diagnosis and prognosis.
  • Stem cells such as leukemia stem cells, LSC
  • CD47 on the surface of tumor cells activates the negative regulatory effect of SHP-1 by combining with SIRP ⁇ on the surface of immune cells such as macrophages and DC cells, and inhibits the migration and phagocytosis of macrophages. This effect is one of the main reasons for the immune escape of tumor cells with high expression of CD47. Blocking the interaction between CD47 on the surface of tumor cells and SIRP on the surface of macrophages by antibodies or other blocking agents can promote the phagocytosis of tumor cells by macrophages and DC cells, further increase the presentation of tumor antigens, and activate adaptation Sexual immunity provides a new idea for the treatment of tumors.
  • Several therapeutic antibodies targeting CD47 are currently in clinical trials. The representative one is Hu5F9.
  • CD47 Although therapeutic antibodies targeting CD47 have shown good therapeutic effects in hematological tumors, they have not progressed smoothly in solid tumors. In addition, because CD47 is widely expressed on the surface of erythrocytes and platelets, it has strong erythrocyte and platelet toxicity, and antigen sedimentation leads to short half-life and large dosage.
  • PD-L1 programmed death ligand-1, which can bind to the receptor PD-1 on the surface of T cells and exert an immunosuppressive effect.
  • PD-L1 is an inhibitory immune checkpoint molecule that is expressed on the cell surface of various malignant tumors such as melanoma, non-small cell lung cancer, renal cell carcinoma, and head and neck squamous cell carcinoma. After PD-L1 binds to the immunosuppressive receptor PD-1 on the surface of T cells, it can induce T cell apoptosis, incapacitation, and exhaustion, thereby inhibiting the activation, proliferation and anti-tumor function of tumor antigen-specific T cells to achieve tumor immune escape.
  • PD-1/PD-L1 blocking antibodies can relieve the immunosuppressive effect of PD-L1 and enhance the recognition and killing of tumor cells by immune cells T cells in the body, thereby achieving the effect of killing tumors.
  • a number of antibody drugs targeting PD-1/PD-L1 have been marketed around the world, which are clinically effective against melanoma, lung cancer, kidney cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, and urothelial carcinoma.
  • a variety of tumors show good therapeutic effects.
  • PD-1/PD-L1 therapeutic antibodies have achieved certain clinical effects, their clinical efficacy is low. %-20%. Therefore, more effective antibody-based drug molecules need to be developed to meet clinical needs.
  • CD47 protein is an immunoregulatory molecule overexpressed on cancer cells, which mainly inhibits the phagocytosis of macrophages by interacting with inhibitory receptor signaling regulatory protein ⁇ (SIRP ⁇ ) and mediates immune escape of various malignant tumors.
  • SIRP ⁇ inhibitory receptor signaling regulatory protein ⁇
  • Bispecific antibodies targeting PD-L1/CD47 can block both CD47 and PD-L1 pathways at the same time, which is expected to obtain stronger anti-tumor activity, thereby solving the problem of low efficacy of PD-L1 antibody alone in clinical practice .
  • the current PD-L1/CD47 bispecific antibody has made some progress in tumor treatment, to a certain extent, it has overcome the defects of using anti-PD-L1 and anti-CD47 alone, but the following defects still exist: 1.
  • Anti-CD47 side In terms of molecular selection, SIRP ⁇ protein is mainly selected as the binding phase, rather than antibody molecules. This antibody-like recombinant protein is far from monoclonal antibody in stability, quality, and PK/PD characteristics. 2.
  • the difference in the sequence structure of the two chains of A and B in the antibody structure brings great difficulties to the expression, purification process and quality control. The mismatch rate of light and heavy chains is high, and the correctly assembled bispecific antibody molecule is difficult to purify and recover. rate is low.
  • the PD-L1/CD47 bispecific antibody still has the potential to bind to erythrocytes to cause hemolysis and agglutination, and it is necessary to further reduce the binding ability to erythrocytes.
  • anti-CD47 humanized antibody and PD-L1/CD47 bispecific antibody need to be further studied.
  • the present invention provides an anti-CD47 humanized antibody and a PD-L1/CD47 bispecific antibody, wherein the anti-CD47 humanized antibody can bind to human CD47, block the binding of CD47 to SIRP ⁇ , and,
  • the antibody is a medium-low affinity antibody against CD47.
  • the antibody only specifically binds to CD47 on the surface of tumor cells, but does not bind to CD47 on the surface of human erythrocytes, thus solving the problems of hemolysis and agglutination caused by the binding of the antibody to erythrocytes.
  • Bispecific antibodies comprise two heavy chains and two light chains, wherein the variable regions of the two heavy chains are heterologous and the variable region sequences of the two light chains are identical, in particular, directed against CD47 and A bispecific antibody to PD-L1, wherein the variable regions of the two heavy chains are derived from the heavy chain variable region of the anti-CD47 monoclonal antibody and the heavy chain variable region of the anti-PD-L1 monoclonal antibody, respectively.
  • the light chain with the same sequence was generated based on the optimized design of the light chain of anti-CD47 monoclonal antibody and the light chain of anti-PD-L1 monoclonal antibody.
  • the bispecific antibodies provided by the present disclosure have light chains with the same sequence, and thus can effectively solve the technical problem of mismatch between light and heavy chains in the bispecific antibody assembly process to a certain extent.
  • a mouse antibody that specifically binds to human CD47 is prepared.
  • an approximate human antibody germline template is selected to prepare a humanized anti-human CD47 monoclonal antibody or a fragment thereof.
  • a humanized anti-human CD47 monoclonal antibody or a fragment thereof which has the activity of blocking the binding of SIRP ⁇ to CD47 on the cell surface and the tumor suppressing activity.
  • the affinity for human CD47 was adjusted by point mutation transformation of the CDRs region and FR region sequence, and the antibody was further differentiated from the surface of tumor cells CD47 and human by light chain mutation.
  • CD47 Binding activity of CD47 on the surface of erythrocytes, a series of anti-CD47 antibodies without erythrocyte binding activity were obtained. Further, the humanized anti-human CD47 mAb was selected to tolerate multiple amino acid site mutations in the light chain CDRs while maintaining the specific binding ability of CD47, thereby providing a new platform for the development of co-light chain bispecific antibodies . in particular:
  • the present invention provides a humanized anti-human CD47 monoclonal antibody or a fragment thereof.
  • a humanized anti-human CD47 monoclonal antibody or a fragment thereof is characterized in that, the humanized anti-human CD47 monoclonal antibody or a fragment thereof comprises: heavy chain CDR1, including X1YX2MX3, wherein X1 is selected from N , S, X2 are selected from V, A, X3 is selected from H, S; Heavy chain CDR2, including YINPX4NX5X6IKYNEKFX7G, wherein X4 is selected from Y, G, X5 is selected from D, E, X6 is selected from G, A, X7 is selected from T , Q; heavy chain CDR3, including EGDFYANYGRLGFX8Y, wherein X8 is selected from A, D; And, light chain CDR1, including RASQDIX9NYLN,
  • its germline templates are selected from IGKV1-33*01
  • the CDRs and/or FR regions of the humanized anti-human CD47 mAb or fragment thereof include point mutations that help reduce or eliminate binding activity to erythrocytes, and at least partially Retain the binding ability of humanized anti-human CD47 mAb or its fragment to tumor cells.
  • the heavy chain CDR1 has the sequence shown in SEQ ID NO: 76, 77, 78 or 87; the heavy chain CDR2 has SEQ ID NO: 79, 80, 81, 82, 83 or 84 The sequence shown; the heavy chain CDR3 has the sequence shown in SEQ ID NO: 85 or 86.
  • the light chain CDR1 has the sequence shown in SEQ ID NO: 46 or 47; the light chain CDR2 has the sequence shown in SEQ ID NO: 50, 51 or 52; the light chain CDR3 Has the sequence shown in SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 68.
  • the humanized anti-human CD47 monoclonal antibody or its fragment described in the humanized anti-human CD47 monoclonal antibody or its fragment has SEQ ID NOs: 8, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22 or 23 shown heavy chain variable region, SEQ region ID NO: 5 shown light chain variable region.
  • the humanized anti-human CD47 monoclonal antibody or the monoclonal antibody of the fragment thereof is a derivatized antibody
  • the derivatized antibody includes CDRs transplantation, affinity maturation, Antibodies obtained by point mutation and chemical modification, wherein the chemical modification includes glycosylation, acetylation, PEGylation, phosphorylation, amidation, protease cleavage, connection with cellular ligands or effector molecules, active reactive groups Regiment protection and/or closure.
  • any of the humanized anti-human CD47 monoclonal antibodies or fragments thereof of the present invention are characterized in that: the humanized anti-human CD47 monoclonal antibodies or fragments thereof include Fab, Fab', F(ab')2 , Fv, scFv or dAb.
  • the present invention provides a humanized anti-human CD47 monoclonal antibody or a fragment thereof, which is based on the mouse-derived anti-human CD47 monoclonal antibody, selecting IGKV1-33*01
  • the heavy chain variable region of the humanized anti-human CD47 monoclonal antibody or its fragment has the sequence shown in SEQ ID NO: 8, and the heavy chain variable region has the sequence of CDRs (HCDR1, HCDR2 , HCDR3), the light chain variable region has the sequence shown in SEQ ID NO: 5, and the light chain variable region has the CDRs sequence (LCDR1, LCDR2, LCDR3).
  • the heavy chain variable region of the humanized anti-human CD47 monoclonal antibody or its fragment is shown in SEQ ID NO:8, and the light chain variable region is shown in SEQ ID NO:5.
  • the humanized anti-human CD47 monoclonal antibody or its fragment has:
  • Heavy chain CDR1 including X 1 YX 2 MX 3 , wherein X 1 is selected from N, S, X 2 is selected from V, A, X 3 is selected from H, S;
  • Heavy chain CDR2 including YINPX 4 NX 5 X 6 IKYNEKFX 7 G, wherein X 4 is selected from Y, G, X 5 is selected from D, E, X 6 is selected from G, A, X 7 is selected from T, Q;
  • the humanized anti-human CD47 monoclonal antibody or its fragment has:
  • Light chain CDR2 including YTSRLX 10 S, wherein X 10 is selected from H, Q, S;
  • the present invention provides a multispecific antibody.
  • the multispecific antibody comprises at least one binding site specific for human CD47, and the specific binding site for human CD47 is obtained from the aforementioned humanized anti-human CD47 monoclonal antibody or Its fragments are provided.
  • the multispecific antibody comprises multiple specific binding sites for different epitopes of human CD47.
  • the multispecific antibody further comprises a specific binding site targeted for non-human CD47.
  • the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.
  • the humanized anti-human CD47 monoclonal antibody or its fragment of the embodiment of the present invention has at least one of the following beneficial technical effects:
  • the humanized anti-human CD47 monoclonal antibody or its fragment of the embodiment of the present invention can specifically bind to CD47 on the cell surface with high affinity, effectively block the interaction between CD47 on the surface of cancer cells and SIRPa, and has a good tumor inhibitory effect ;
  • the affinity of the antibody to human CD47 was adjusted by point mutation transformation of the CDRs region and FR region sequence, and the antibody was further differentiated from the surface CD47 of tumor cells through light chain mutation. Binding activity to CD47 on the surface of human erythrocytes to obtain a series of anti-CD47 antibodies with high binding activity to CD47 on the surface of tumor cells and low binding activity to erythrocytes, effectively solving the clinical problem of anti-CD47 antibodies due to the combination of red blood cells and platelets. The antibody concentration is low, which leads to the problem of high clinical dosage, and significantly reduces the toxic and side effects of anti-human CD47 antibody. Molecular selection.
  • the humanized anti-human CD47 monoclonal antibody of the present invention can tolerate multiple amino acid site mutations in the variable region of the light chain under the condition of maintaining the specific binding ability of CD47, so as to be used for the development of co-light chain bispecific antibodies Provide new platforms.
  • the present invention provides a method of preparing a bispecific antibody. According to an embodiment of the present invention, the method includes:
  • the heavy and light chains of the hybrid antibody comprise the heavy chain variable region of the first antibody and the light chain variable region of the second antibody, respectively.
  • site mutation is performed on the hybrid antibody, an antibody hybrid group is constructed, the binding ability of each antibody to the first antigen is detected, and the first heavy chain is selected from the first heavy chain.
  • the hybrid antibody formed with the light chain of the second antibody has high affinity for the first antigen and good specificity.
  • each amino acid residue position of the variable region of the light chain mutant of the second antibody is at least the amino acid residue of the corresponding position of the variable region of the light chain of the first antibody molecule , and/or the amino acid residues of the corresponding positions in the variable region of the light chain of the second antibody molecule are the same.
  • step (5) if there is a difference between the common light chain variable region selected in step (4) and the light chain variable region of the second antibody, in step (5), according to the heavy chain variable region of the second antibody Selecting the second heavy chain variable region includes: constructing a second antibody heavy chain variable region mutant population and co-expressing it with the common light chain variable region to prepare second antibody heavy chain variable region mutants/common light chain The variable region hybrid antibody is tested for its binding ability to the second antibody, and the second heavy chain is selected;
  • the second heavy chain variable region selected according to the heavy chain variable region of the second antibody in step (5) is the second The heavy chain of the antibody serves as the second heavy chain.
  • the present invention provides a bispecific antibody or antigen-binding fragment thereof.
  • the bispecific antibody or antigen-binding fragment thereof comprises two heavy chains and two light chains, wherein the first heavy chain variable region is derived from the heavy chain variable region of an anti-CD47 monoclonal antibody , It can form a CD47 binding site with the light chain variable region, and the second heavy chain variable region is derived from the heavy chain variable region of the anti-PD-L1 monoclonal antibody, which can form PD-L1 with the light chain variable region Binding site; wherein, the sequences of the light chain variable regions of the two light chains are the same, and are selected from the light chain of an anti-CD47 monoclonal antibody, the light chain of an anti-PD-L1 monoclonal antibody, and a sequence intermediate in structure.
  • the light chain variable region of the first antibody molecule and the light chain variable region of the second antibody molecule belong to the same antibody light chain germline, and have the same or highly similar framework regions (FR) .
  • the common light chain variable region has amino acid residues common between the light chain variable region of the first antibody molecule and the light chain variable region of the second antibody molecule, and the light chain variable region of the first antibody molecule has the same amino acid residues.
  • the first heavy chain variable region of the bispecific antibody or its antigen-binding fragment is obtained by point mutation on the basis of the heavy chain variable region of the first antibody molecule, and the first heavy chain variable region is obtained by point mutation.
  • a heavy chain variable region at least partially retains the ability of the heavy chain variable region and light chain variable region of the first antibody molecule to form a first antigen binding site.
  • the present invention also provides a bispecific antibody or an antigen-binding fragment thereof targeting PD-L1 and CD47.
  • the bispecific antibody or antigen-binding fragment thereof comprises two heavy chains and two light chains, wherein the first heavy chain variable region is derived from the heavy chain variable region of an anti-CD47 monoclonal antibody , It can form a CD47 binding site with the light chain variable region, and the second heavy chain variable region is derived from the heavy chain variable region of the anti-PD-L1 monoclonal antibody, which can form PD-L1 with the light chain variable region Binding site; wherein, the sequences of the light chain variable regions of the two light chains are the same, and the light chain variable regions are based on the light chain of the anti-CD47 monoclonal antibody and the anti-PD-L1 monoclonal antibody.
  • variable region of the light chain is matched with the variable region of the first heavy chain to form a first variable region binding site and specifically binds to CD47, and the variable region of the light chain is combined with the variable region of the first heavy chain.
  • the second heavy chain variable region is matched to form a second variable region binding site and specifically binds to PD-L1, wherein the anti-CD47 monoclonal antibody is the aforementioned humanized anti-human CD47 monoclonal antibody or its Fragment.
  • the first variable region binding site specifically binds to CD47, and its affinity is similar to that of the original CD47 mAb variable region and CD47, and the first variable region binding site It specifically binds to PD-L1, and its affinity is similar to that of the variable region of the original PD-L1 mAb and that of PD-L1.
  • the anti-PD-L1 monoclonal antibody comprises a heavy chain variable region shown in SEQ ID NO:3 and a light chain variable region shown in SEQ ID NO:1.
  • the first heavy chain comprises a heavy chain variable selected from the group consisting of SEQ ID NOs: 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 Area.
  • the light chain variable region has the sequence shown in SEQ ID NO: 1, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34.
  • the antibodies or antigen-binding fragments thereof include, but are not limited to, complete antibodies, F(ab')2.
  • the complete antibody is of IgG1 or IgG4 type.
  • the Fc segments of the two heavy chains comprise a "knobs-into-holes" mutation
  • the Fc segment of the first heavy chain comprises the H315R mutation
  • the Fc segment of the other second heavy chain comprises the K319E mutation
  • the amino acid mutation sites of the Fc segment use Kabat's Eu numbering system.
  • the present invention provides a polynucleotide.
  • the polynucleotide encodes the aforementioned humanized anti-human CD47 monoclonal antibody or a fragment thereof, or the aforementioned multispecific antibody, or the aforementioned bispecific antibody targeting PD-L1 and CD47, or its antigen-binding fragment.
  • the present invention provides a carrier.
  • the vector includes the aforementioned polynucleotide.
  • the present invention provides a host cell.
  • the host cell comprises the aforementioned polynucleotide or the aforementioned vector.
  • the present invention provides a method for preparing an antibody or antigen-binding fragment thereof.
  • the method comprises the following steps: (1) culturing the host cell of claim 21 under conditions suitable for expressing the bispecific antibody or its antigen-binding fragment; (2) separating and purifying from the cell culture Bispecific antibodies or fragments thereof.
  • the technical solution of the bispecific antibody or antigen-binding fragment thereof targeting PD-L1 and CD47 according to the embodiment of the present invention has at least one of the following advantages:
  • the bispecific antibodies prepared in the examples of the present invention have dual binding arms of natural antibodies, and each binding arm has a complete Fab structure, avoiding the introduction of receptor chains (such as CD47 receptor SIRP ⁇ ) into the antibody molecule.
  • receptor chains such as CD47 receptor SIRP ⁇
  • the disadvantages of non-antibody components such as structural instability and mutual interference of functions.
  • the regulation of the binding ability of the two binding arms is realized, for example, the first antigen/antigen epitope is prepared.
  • the effect of the three CDRs of the heavy chain is greater than that of the three CDRs of the light chain
  • the effect of CDR3 is greater than that of CDR2 and CDR1.
  • the design of the common light chain not only simplifies the recombinant expression method, but also completely eliminates the problem of light and heavy chain mismatch during the recombinant expression of the bispecific antibody, which significantly simplifies the production process and improves the product qualification rate and uniformity.
  • "knobs-into-holes" mutations can also be introduced in the Fc segment to enhance the accuracy of the pairing of the Fc segment of the bispecific antibody heavy chain and avoid homologous heavy chain dimerization.
  • the bispecific antibody has moderate anti-CD47 activity by introducing a slight difference in sequence structure at the antigen-binding site of the above-mentioned anti-CD47, which not only maintains the binding activity of the bispecific antibody to CD47 on the surface of tumor cells, but also enhances the anti-CD47 activity of the bispecific antibody.
  • the tumor suppressor effect of PD-L1 activity in turn, the binding to CD47 on the surface of erythrocytes is significantly reduced, which not only reduces or eliminates undesired consequences such as hemolysis and hemagglutination, but also reduces the amount of systemic administration.
  • the present invention provides a composition.
  • the composition comprises the aforementioned humanized anti-human CD47 monoclonal antibody or a fragment thereof, the aforementioned multispecific antibody, the aforementioned bispecific antibody targeting PD-L1 and CD47, or antigen binding thereof At least one of the fragment, the aforementioned polynucleotide, the aforementioned vector, and the aforementioned host cell.
  • the present invention provides the aforementioned humanized anti-human CD47 monoclonal antibody or fragment thereof, the aforementioned multispecific antibody, the aforementioned bispecific antibody targeting PD-L1 and CD47, or an antigen thereof Use of the binding fragment, the aforementioned polynucleotide, and the aforementioned composition in the preparation of a medicament for treating tumors and/or improving the immune response of the body.
  • the tumors include melanoma, lung cancer, kidney cancer, Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, acute and chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma cancer, bladder cancer, ovarian cancer, breast cancer, rectal cancer, prostate cancer, kidney cancer and multiple myeloma.
  • the present invention provides a method of treating a subject having a tumor.
  • the method comprises administering to the subject a therapeutically effective amount of a composition, which is the aforementioned composition.
  • PD-L1 the full name of PD-L1 (programmed death ligand 1), is the full name of programmed death receptor ligand 1, also known as surface antigen differentiation cluster 274 (cluster of differentiation 274, CD274) or B7 homologue (B7 homolog 1, B7-H1), encoded by the CD274 gene, is a ligand for PD-1 (programmed cell death 1, programmed cell death 1).
  • PD-L1 is a type 1 transmembrane protein with a size of 40kDa, which is expressed on T cells, B cells and other immune cells and tumor cells.
  • the immune system will react to foreign antigens accumulated in the lymph nodes or spleen, triggering Antigen-specific cytotoxic T cells (CD8+Tcell proliferation).
  • CD8+Tcell proliferation Antigen-specific cytotoxic T cells
  • PD-L1 on the tumor cell membrane binds to PD-1 on immune cells such as T cells
  • tumor cells send inhibitory signals to reduce the proliferation of CD8+ T cells in lymph nodes, which in turn causes T cells to fail to recognize tumor cells and respond to tumor cells. Killing effect, the body's immune function is inhibited.
  • CD47 also known as integrin-related protein, has a molecular weight of about 50 kDa and is expressed in both normal cells and tumor cells. CD47 can bind to the protein SIRP ⁇ on macrophages to provide a "don't eat me” signal, thereby inhibiting the phagocytic function of macrophages and leading to immune escape of tumors. In tumor cells, senescent red blood cells and platelets, blocking the CD47-SIRP ⁇ interaction triggers phagocytosis. On the one hand, according to this mechanism of action, CD47 has gradually increased in tumor immunotherapy in recent years and is a potentially effective and widely used target for tumor immunotherapy.
  • the term "specificity” refers to determining the presence or absence of a protein and/or other biologically heterogeneous population that, under specified conditions, binds a specific ligand/antigen to a specific receptor/antibody and does not Binds to other proteins present in the sample in significant amounts.
  • antibody herein is intended to include full-length antibodies and any antigen-binding fragments (ie, antigen-binding portions) or single chains thereof.
  • Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, namely CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs) separated by more conserved framework region (FR) regions.
  • CDRs complementarity determining regions
  • FR conserved framework region
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the traditional complement system.
  • monoclonal antibody or “monoclonal antibody” or “monoclonal antibody composition” refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are composed to exhibit a single binding specificity and affinity for a particular epitope.
  • an "antigen-binding fragment” (or simply an antibody portion) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.
  • binding fragments included in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1; (ii) F(ab')2 fragments, comprising hinge region II Bivalent fragment of two Fab fragments linked by a sulfur bridge; (iii) Fd fragment composed of VH and CH1; (iv) Fv fragment composed of antibody one-arm VL and VH; (v) dAb fragment composed of VH ( Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) Nanobodies, a single variable domain and two constant domain heavy chain variable region.
  • the two domains of the Fv fragment, VL and VH are encoded by different genes, they can be linked by recombinant methods via a synthetic linker that makes the two into a single protein chain in which the VL and VH domains pair to form a monovalent molecule (called Single chain Fc (scFv); see eg Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • scFv Single chain Fc
  • These single chain antibodies are also intended to be included within the meaning of the term.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be screened for function in the same manner as intact antibodies.
  • Antigen-binding fragments of the present invention include those capable of specifically binding antigenic molecules.
  • Examples of antibody binding fragments include, for example, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single chain Fv (scFv) fragments, and single domain fragments.
  • Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab' fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F(ab')2 pepsin digest. Additional chemical conjugation of antibody fragments is known to those of ordinary skill in the art.
  • Fab and F(ab')2 fragments lack the crystallizable (Fc) region of intact antibodies, are cleared more rapidly from the animal's circulation, and may have less nonspecific tissue binding than intact antibodies (see e.g., Wahl et al. Man, 1983, J. Nucl. Med. 24:316).
  • an "Fc” region is a fragment crystallizable constant region of an antibody that does not contain an antigen-specific binding region.
  • the Fc region consists of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody.
  • the IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3 and CH4 domains) in each polypeptide chain.
  • Human antibodies include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from human immunoglobulin libraries or animals that are transgenic for one or more human immunoglobulins, and Endogenous immunoglobulins are not expressed. Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See US Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741.
  • Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but that express human immunoglobulin genes.
  • ⁇ ,PCT ⁇ WO 98/24893;WO 92/01047;WO 96/34096;WO 96/33735; ⁇ 5,413,923;5,625,126;5,633,425;5,569,825;5,661,016;5,545,806;5,814,318;5,885,793;5,916,771; ⁇ 5,939,598 ⁇
  • companies such as LakePharma, Inc. (Belmont, CA) or Creative BioLabs (Shirley, NY) can engage in providing human antibodies against selected antigens using techniques similar to those described above.
  • Fully human antibodies that recognize epitopes of choice can be generated using a technique known as "guided selection.”
  • selected non-human monoclonal antibodies eg mouse antibodies
  • guided selection is used to guide the selection of fully human antibodies recognizing the same epitope (see, Jespers et al., 1988, Biotechnology 12:899-903).
  • the heavy chain (heavy chain, H chain) is about twice the size of the light chain, contains 450-550 amino acid residues, and has a molecular weight of about 55 or 75 kD.
  • Each H chain contains a cyclic peptide composed of 4 to 5 intrachain disulfide bonds. Different H chains have different antigenicity due to the arrangement sequence of amino acid composition, the number and position of disulfide bonds, and the types and quantities contained.
  • H chain antigenicity According to the difference in H chain antigenicity, they can be divided into 5 categories: ⁇ chain , ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain, different H chain and L chain ( ⁇ or ⁇ chain) make up the complete immunoglobulin molecules are called IgM, IgG, IgA, IgD and IgE respectively.
  • the ⁇ , ⁇ and ⁇ chains contain 4 peptides, and the ⁇ and ⁇ chains contain 5 cyclic peptides.
  • light chain refers to a polypeptide chain that is smaller in molecular weight relative to the heavy chain in an immunoglobulin monomer molecule.
  • the variable region (VL) of the amino acid composition sequence in the region near the amino terminal (N-terminal) 1/2 of each light chain is the light chain variable region (VL), which is an integral part of the Ig molecule and the antigen binding site.
  • the amino acid composition and arrangement sequence in the remaining half of the region are relatively stable in the light chain constant region (CL). Due to some differences in the amino acid sequence within the constant region of the light chain, there are two types of light chains, ⁇ and ⁇ .
  • germline also known as the germline, is the antibody-forming cell that has the complete set of genes encoding Ig molecules (i.e. a limited number of C genes and an unknown number of V genes, which are formed through long-term evolution and are derived from parental cells through germ cells) Passed on to offspring, the heavy chain gene of human immunoglobulin is encoded by V-D-J-C gene fragment; the light chain gene is encoded by V-J-C gene fragment, and the number of gene fragments varies.
  • the human heavy chain gene is located on chromosome 14
  • the long arm spanning about 1,100kb, is composed of four gene fragments V, D, J and C, including about 95 Va gene fragments (divided into seven families of VH1 ⁇ VH7, of which 65 functional gene fragments and 27 D genes) fragment, 6 JH gene fragments and 9 CH gene fragments.
  • the Va gene fragment is located upstream
  • the D gene fragment is located between the VH and JH gene clusters
  • the JH gene is located downstream of DH, and is separated from the downstream C gene region by about 7Kb.
  • Cluster arrangement spanning about 200kb, P and S genes are located immediately downstream of the JH gene segment, and C8 downstream is followed by C ⁇ , C ⁇ and C ⁇ .
  • Human light chain genes are divided into ⁇ and x genes, which are located on chromosome 22, respectively. Arm and short arm of chromosome 2. There are about 40 functional VK gene fragments, 5 functional J and 1 C ⁇ after the Vc gene fragment; about 30 V ⁇ gene fragments, 4 J ⁇ gene fragments and 4 C ⁇ genes Fragment.
  • EC50 also known as half-maximal effect concentration, refers to the concentration of antibody that elicits 50% of the maximal effect.
  • bispecific antibodies an antibody structure that can bind to different epitopes on the same or different antigens.
  • bispecific antibodies are capable of bridging two different molecules, acting to recruit effector molecules, effector cells, viruses, and drug carrier systems to target structures.
  • bispecific antibodies of IgG-like structure are usually expressed in single cells, but the antibody is composed of 2 light (L) and 2 heavy (H) chains and is co-expressed in a single cell line
  • L light
  • H heavy
  • Purifying a sufficient amount of target antibody from a complex system containing nearly 10 mixtures is extremely challenging, resulting in problems with low product homogeneity and yield, and complicated subsequent purification processes.
  • the production strategy of the marketed product Catumaxomab (antibody type is Triomab) has been improved on this basis.
  • Triomab uses rat hybridoma and mouse hybridoma for somatic fusion, and its species-restricted H-L chain pairing (Species restricted H-Lchain pairing) strategy can reduce the proportion of random light and heavy chain mismatches ( ⁇ 10%).
  • species-restricted H-L chain pairing species restricted H-Lchain pairing
  • the target with both rat Fc segment and mouse Fc segment can be isolated by one-step protein A affinity chromatography purification antibodies, thus simplifying purification steps.
  • bispecific antibodies constructed based on Triomab technology are murine antibodies and have strong immunogenicity, so their clinical application is limited.
  • Figure 1 Schematic diagram of the affinity of Hz140 and CD47-his determined by Fortebio;
  • Figure 2 Schematic diagram of the results of FACS analysis of the binding activity of hz140 to tumor cells U937;
  • Figure 3 Schematic diagram of the results of FACS analysis of the binding activity of hz140 to CCRF in tumor cells
  • Figure 4 Schematic diagram of the results of FACS analysis of the binding activity of hz140 to erythrocytes
  • Figure 5 Schematic diagram of the results of FACS analysis of hz140 on the blocking activity of SIRPa and CCRF cell surface CD47;
  • Figure 6 FACS analysis of the results of hz140 blocking activity of CD47 on the surface of SIRPa and U937 cells;
  • Figure 7 Schematic diagram of the observation results of hz140 on the survival time of CCRF-SB leukemia model mice
  • Figure 8 Schematic diagram of the in vivo efficacy observation results of anti-CD47 antibody on human xenografted lymphoma Raji model
  • Figure 9 Schematic diagram of the quantitative results of the signal intensity quantification of the anti-tumor efficacy of 2MW1531 on the immunodeficient mouse tail vein xenograft Raji blood tumor model;
  • Figure 10 Schematic diagram of the amino acid sequence alignment of hz140 light chain and hz182 light chain
  • Figure 11 Schematic diagram of the analysis results of the binding activity of 2MW1531 to MC38-hPD-L1/hCD47 cells;
  • Figure 12 Schematic diagram of the analysis results of the binding activity of 2MW1531 to A431 cells
  • Figure 13 Schematic diagram of the analysis results of the binding activity of 2MW1531 to human erythrocytes
  • Figure 14 Schematic diagram of the results of in vitro hemagglutination test with anti-human CD47/PD-L1 bispecific antibody
  • Figure 15 Schematic diagram of the experimental results (tumor volume) of the antitumor efficacy of 2MW1531 on hPD-L1/hCD47/hSIRP ⁇ transgenic mice subcutaneously transplanted with MC38-hPDL1 mouse colon cancer tumor model;
  • Figure 16 In vivo imaging image of 2MW1531 for the detection of anti-tumor efficacy of immunodeficient mouse tail vein xenograft Raji blood tumor model.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as “first” or “second” may expressly or implicitly include one or more of that feature. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.
  • Example 1 Preparation of anti-CD47 high-affinity antibody 140 and control antibody
  • Hybridoma production was performed by immunizing Balb/c mice with CD47-mFc. Serum antibody titers were detected 34 days or 57 days after the primary immunization, respectively. Select the mice whose serum titer meets the requirements of the fusion experiment (serum antibody titer: 1:102400) spleen cells and myeloma cells FO in an appropriate ratio (5:1) to fuse under the action of a fusion agent to prepare hybridomas Monoclonal cells.
  • Hybridoma cells were cultured in selective medium R1640-HAT, and 651 hybridoma supernatant samples were detected by ELISA on days 10-14, including cell binding activity (FACS), CD47/SIPR blockade , and the cross-reactivity with recombinant cyno-CD47-mFc, clone 140 was screened.
  • the above cloned light and heavy chain variable region genes were obtained, and the light and heavy chain variable region genes were cloned into a recombinant expression vector containing human C ⁇ and IgG4 gene reading frames, and transiently expressed on HEK293 cells.
  • Analysis, clone 140 murine antibody light and heavy chain variable region amino acid sequences are SEQ ID NO: 43 and SEQ ID NO: 44, respectively.
  • SEQ ID NO.43 Murine 140 light chain variable region amino acid sequence
  • SEQ ID NO.44 Murine 140 heavy chain variable region amino acid sequence
  • the following antibodies were prepared as reference antibodies, all in the form of IgG4.
  • the recombinant expression strategy of the control antibody was realized by conventional molecular biology methods, and expression and purification were completed by transient expression of HEK293.
  • Forty Seven's clinical research antibody hu5F9 US2015/0183874A1, hu5F9V2, light and heavy chain variable region sequences: SEQ ID NO.35 and SEQ ID NO.36
  • I-Mab's anti-CD47 antibody 1F8 CN 110582515 A, light and heavy chain variable region SEQ ID NO. 37 and SEQ ID NO. 38).
  • SEQ ID NO.36 hu5F9 heavy chain variable region amino acid sequence
  • SEQ ID NO.8 140 Humanized heavy chain variable region amino acid sequence
  • SEQ ID NO.5 140 Light chain variable region amino acid sequence after humanization
  • hz140 inhibits the blocking activity of hz140 was basically comparable to that of hu5F9; the blocking activity of hz140 was significantly stronger than that of hu5F9 on CCRF cells with relatively low CD47 abundance.
  • mice Balb/c nude mice, 6-8 weeks old, were injected intraperitoneally with cyclophosphamide 2mg/mice/200 ⁇ l for immunosuppression, and 48 hours later, freshly treated CCRF-SB cells 6.25 ⁇ 106 were inoculated into the tail vein.
  • D5 Five days after inoculation (D5), the body weight of the mice was measured, and the mice were randomly assigned to each experimental group according to the body weight data, with 8 mice in each group.
  • Different doses of test antibodies i.v., twice a week, 6 times in total
  • the positive control antibody hu5F9 can prolong the survival of the model mice, but the difference between the high-dose group (10mg/kg) and the low-dose group (3.3mg/kg) of hu5F9 No significant difference.
  • hz140 not only significantly prolonged the survival of model mice compared with the negative control, but also had a better effect than the positive control antibody hu5F9 at the same dose.
  • the effect of hz140 high-dose group was better than that of hz140 low-dose group, suggesting that hz140 showed a dose-dependent effect on prolonging the survival time of model mice.
  • the heavy chain CDRs sequence was mutated and designed to reduce the Koff value of the antibody binding to CD47 in order to reduce its impact on RBC and PLT. .
  • a number of humanized hz140 heavy chain mutants were constructed, amino acid site-directed mutagenesis was performed on the humanized hz140 heavy chain, and the mutant heavy chain was paired with the humanized hz140 light chain for expression, and the affinity was determined.
  • the comparison results are as follows shown in Table 2. And further mutate on the basis of hm10 to obtain the final sequence of 140Hm13 as shown in SEQ ID NO.23.
  • mutants were designed and combined with h140L (SEQ ID NO. 5) and the similar light chain sequence hz182L (SEQ ID NO. 1) of another heterologous antibody, respectively, and the mutants were evaluated for their interaction with the tumor cell surface. Differences in binding of CD47 to erythrocyte surface CD47.
  • the light chain variable region sequence of hz140 was subjected to mutation design, and the mutant design was shown in Table 3 below; the light chain (h182L) of hz182 was subjected to mutation design, and the mutant design was shown in Table 4 below.
  • the binding of different mutants to tumor cells (1 ⁇ g/mL) and erythrocytes (150 ⁇ g/mL) was evaluated by FACS, and mutation sites with binding differences were found.
  • SEQ ID NO.1 Light chain sequence hz182L of humanized 182 antibody
  • the hz140 heavy chain mutant h140Hm13 was co-expressed with h140 light chain mutants (h140Lm1 ⁇ h140Lm18) and h182 light chain mutants (hz182-Lm1 ⁇ hz182-Lm11) to prepare recombinant antibodies. Anti-CD47-specific antibody without specific binding.
  • mutant results of h140L and h182L in Table 3 and Table 4 it was found that the erythrocyte binding ability of h140Lmut8-mut10 was significantly reduced, and the tumor binding activity was reduced, but still maintained a certain binding activity. Especially for mut8 and mut10, the erythrocyte binding ability was completely lost, but the tumor cell binding ability was maintained.
  • the reverse mutation of h182L also confirmed that the erythrocyte binding activity was restored by mutating h182L to h182Lmut8 (Y to R).
  • the anti-human PD-L1 humanized antibody was prepared according to the previous patent application (patent application number: CN201911419802.5).
  • the amino acid sequence of the light chain variable region (hz182-L) of the humanized antibody is shown in the sequence SEQ ID NO.1, and the coding nucleotide sequence is shown in the sequence SEQ ID NO.2; the heavy chain variable region (hz182-H) amino acid sequence
  • the sequence is shown in sequence SEQ ID NO.3
  • the coding nucleotide sequence is shown in sequence SEQ ID NO.4.
  • the light chain variable region (hz140-L) amino acid sequence of hz140 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6; the light chain constant region amino acid sequence See SEQ ID NO.7; heavy chain variable region (hz140-H) amino acid sequence see SEQ ID NO.8, nucleotide sequence see SEQ ID NO.9; heavy chain constant region amino acid sequence see SEQ ID NO.10.
  • the light chain of hz182 (hz182-L) and the heavy chain of hz140 (hz140-H) were combined for expression, and the expression supernatant was subjected to affinity purification to obtain the anti-human CD47 antibody hz140-H/hz182- L.
  • the affinity of the antibody was measured using the Octet QKe instrument of Fortebio Company, the antibody was immobilized on the sensor surface (AHC Sensor) of the anti-human Fc capture antibody, and reacted with the CD47 extracellular domain recombinant antigen in the liquid phase (purchased from Yiqiao Shenzhou, Cat: 12283-H08H; working concentration of 100 nM), and kinetic parameters were determined.
  • the results showed that hz140-H-/hz182-L had good binding activity to CD47 recombinant protein (Table 5).
  • Example 7 Preparation of anti-CD47 antibody heavy chain mutant/anti-PD-L1 antibody light chain hybrid antibody (hz140-Hm/hz182-L)
  • the heavy chain of the hybrid antibody hz140-H/hz182-L was genetically engineered and the affinity of the recombinant antibody for CD47 was assessed. Specifically, by analyzing the distribution frequency of amino acid species at each site in the variable region of the antibody, rationally design mutations at the amino acid sites that may affect the affinity of the antibody, and carry out the combination of different mutation sites; then the mutation is introduced into the antibody by site-directed mutagenesis PCR The corresponding site of the coding gene is used to construct a mutant expression plasmid. Finally, the mutated heavy chain hz140-Hm and light chain hz182-L were combined for expression, and the expression supernatant was subjected to affinity purification.
  • the affinity between the obtained mutant antibody and CD47 recombinant protein was determined by Fortebio, and the antibody was immobilized on the sensor surface (AHC Sensor) of the anti-human Fc capture antibody, and reacted with the CD47 extracellular region recombinant antigen in the liquid phase (working concentration is 100 nM), the binding time and the dissociation time were both 300 seconds, and the kinetic parameters were determined.
  • the mutation site of the heavy chain hz140-H and the affinity of the mutant hz140-Hm to recombinant CD47 are shown in Table 6.
  • Example 8 Preparation of anti-CD47 antibody heavy chain mutant 13/anti-PD-L1 antibody light chain mutant hybrid antibody (hz140-Hm13/hz182-Lm)
  • the light chain of the hybrid antibody hz140-Hm13/hz182-L was genetically engineered and the affinity of the recombinant antibody for CD47 was assessed.
  • the design principles of the light chain mutants are as follows: keep the common amino acid residues between the light chain of the hz140 antibody and the light chain of the hz182 antibody, mutate the amino acid residues with differences, and the mutated amino acid residues are preferentially the light chain of hz140 antibody or the light chain of hz182 antibody
  • the amino acid at this position of the light chain can also be other types of amino acids.
  • Table 7 shows the light chain hz182-L mutation site and the affinity of the mutant hz182-Lm to recombinant CD47.
  • the coding genes of the light and heavy chain variable regions of the anti-CD47 control antibodies hu5F9 (publication number: US 9017675B2) and 1F8 (publication number: CN 110582515 A) were synthesized, and the light and heavy chain variable region coding genes were cloned respectively
  • the operation instructions of the reagent 293fectin, the light and heavy chain plasmids of the control antibody were transferred into HEK293 cells for recombinant expression. 5-6 days after the cells were transfected, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography to obtain a control antibody.
  • the light chain variable region amino acid sequence of the control antibody hu5F9 is shown in SEQ ID NO.35
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO.36.
  • the light chain variable region amino acid sequence of the control antibody 1F8 is shown in SEQ ID NO.37
  • the heavy chain variable region amino acid sequence is shown in SEQ ID NO.38.
  • the expression vector of the two heavy chains and the expression vector of the common light chain of the two antibodies or their mutants were simultaneously transferred into eukaryotic cells for expression, and a bispecific antibody in the form of co-light chain "knobs-into-holes" was obtained by affinity purification, named 2MW1531-p And 2MW1531-m1 ⁇ m11.
  • the mutant information is shown in the table below.
  • the Fc mutation site of 2MW1531 hz140-H is: H315R, the mutated constant region amino acid sequence (named hz140-H-knobs, A chain Fc) is shown in SEQ ID NO.39, and the hz140-H-A amino acid sequence is shown in SEQ ID NO. 40.
  • the Fc mutation site of hz182-H is: K319E, the mutated constant region amino acid sequence (named hz182-H-holes, B chain Fc) is shown in SEQ ID NO.41; hz182-H-B amino acid sequence is shown in SEQ ID NO. .42.
  • the light and heavy chain matching information is shown in Table 8.
  • the binding activity of 2MW1531-p and its mutants to human erythrocytes and tumor cells was analyzed by FACS.
  • the selected antibody concentration was 1000 nM; in the experiment of analyzing the binding to tumor cells, the selected antibody concentration was 100 nM.
  • Table 10 The analysis results are shown in Table 10 below. 2MW1531-p and most of the mutants did not bind or weakly bind to human erythrocytes, but exhibited good binding activity to tumor cells.
  • Example 10 Analysis of the binding activity of anti-CD47/PD-L1 bispecific antibody 2MW1531 to cell surface antigens
  • the binding activity of 2MW1531(m9) was analyzed by flow cytometry using the mouse melanoma cell line MC38-hPD-L1/hCD47, which was humanized with both CD47 and PD-L1.
  • the results are shown in Figure 11.
  • the results show that the binding ability of the anti-human CD47/PD-L1 bispecific antibody 2MW1531 to the mouse melanoma cell line MC38-hPD-L1/hCD47 expressing human CD47 and human PD-L1 is high.
  • Flow cytometry was used to analyze the binding activity of human skin squamous cell carcinoma cell line A431 expressing both CD47 and PD-L1 to 2MW1531(m9).
  • the binding ability of the human skin squamous cell carcinoma cell line A431, the anti-human CD47/PD-L1 bispecific antibody 2MW1531 was higher than that of the anti-PD-L1 mAb, and the control anti-CD47 mAb 1F8.
  • Example 11 Binding activity of anti-human CD47/PD-L1 bispecific antibody 2MW1531 to erythrocyte surface antigen
  • Example 12 Anti-human CD47/PD-L1 bispecific antibody 2MW1531 in vitro RBC agglutination assay
  • the results are shown in Figure 14.
  • the experimental results show that in the agglutination test results of human erythrocytes, CD47 humanized C57 mouse erythrocytes, and monkey erythrocytes, anti-CD47 mAbs 5F9 and 1F8 have no erythrocyte agglutination, while 2MW1531 and NC-IgG4 All erythrocytes were concentrated at the bottom of the microplate without reticular agglutination.
  • the above results show that 2MW1531 has no red blood cell aggregation or hemolysis at the current concentration.
  • Example 13 Anti-human CD47/PD-L1 bispecific antibody 2MW1531 anti-tumor activity in vivo
  • mice Female hPD-L1/hCD47/hSIRP ⁇ transgenic mice (C57-derived hPD-L1/hCD47/hSIRP ⁇ transgenic mice) right Subcutaneously in the lateral anterior flank, when the tumor grows to about 50-100mm3, the tumor is divided into groups and intraperitoneally administered with 2MW1531 (m7) or isotype control IgG at a dose of 20mg/kg, 10mg/kg, twice a week. The tumor volume and body weight were measured at the same time of each administration, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded.
  • 2MW1531 m7
  • IgG isotype control IgG
  • the in vivo imaging diagram in Figure 16 shows that in the mouse model of Raji tumor cell transplantation without PD-L1 expression, the anti-human CD47/PD-L1 bispecific antibody 2MW1531 has an effect on Raji tumor cells.
  • the growth has a significant inhibitory effect, and the inhibitory effect on the proliferation of Raji tumor cells is comparable to that of the anti-CD47 monoclonal antibody 5F9;
  • Figure 9 shows the results of quantitative analysis of the radiation signal, after intravenous inoculation of Raji tumor cells, 2MW1531 was used for intraperitoneal injection treatment, weekly 2 times, it can significantly inhibit the growth of tumor cells, and the therapeutic effect is comparable to that of the anti-CD47 monoclonal antibody 5F9.

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Abstract

提供了抗体及其制备方法，抗体诸如人源化抗人CD47单克隆抗体或其片段和靶向PD-L1和CD47的双特异性抗体或其抗原结合片段，其中，双特异性抗体包括两条重链和两条轻链，其中所述两条重链的可变区是异源的，所述两条轻链可变区序列相似。提供的双特异性抗体，具有相似序列的轻链，因而克服了双特异性抗体装配过程中轻重链错配的技术问题。

Description

抗体及其制备方法
优先权信息
本申请请求2020年11月12日向中国国家知识产权局提交的、专利申请号为202011260418.8的专利申请的优先权和权益，以及2020年11月13日向中国国家知识产权局提交的、专利申请号为202011269292.0的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明属于抗体工程领域，具体涉及抗体及其制备方法，更具体地，涉及人源化抗人CD47单克隆抗体或其片段、多特异性抗体、靶向PD-L1和CD47的双特异性抗体或其抗原结合片段、多核苷酸、载体、宿主细胞、制备抗体或其抗原结合片段的方法和组合物及其相关应用。
背景技术
CD47在大部分肿瘤细胞中高表达，并且它可以作为肿瘤诊断及判断预后的一个标准。如在急慢性髓系白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤以及膀胱癌、卵巢癌、乳腺癌、直肠癌、前列腺癌、肾癌、多发性骨髓瘤等多种实体肿瘤细胞或肿瘤干细胞(如白血病干细胞leukemia stem cells，LSC)存在过表达，并且其高表达与临床预后差相关。研究表明，肿瘤细胞表面的CD47通过与巨噬细胞、DC细胞等免疫细胞表面的SIRPα结合，激活SHP-1的负向调节效应，抑制巨噬细胞的迁移及吞噬作用。该作用是高表达CD47的肿瘤细胞发生免疫逃逸的主要原因之一。通过抗体或其他阻断剂阻断肿瘤细胞表面的CD47与巨噬细胞表面的SIRP的相互作用，可以促进巨噬细胞、DC细胞对肿瘤细胞的吞噬，并进一步增加肿瘤抗原的提呈，激活适应性免疫，为肿瘤的治疗提供了新的思路。目前有多个靶向CD47的治疗性抗体处于临床实验阶段。其中具有代表性的是Hu5F9。靶向CD47的治疗性抗体在血液肿瘤上虽然表现出良好的治疗效果，但是在实体瘤上进展并不顺利。此外，由于CD47广泛表达于红细胞和血小板表面，因此具有较强的红细胞和血小板毒性，并且抗原沉降导致半衰期短，用药量大。
PD-L1全称是程序性死亡配体-1，可以结合T细胞表面的受体PD-1，发挥免疫抑制作用。PD-L1属于抑制型免疫检查点分子，表达于黑色素瘤、 非小细胞肺癌、肾细胞癌、头颈部鳞癌等多种恶性肿瘤细胞表面。PD-L1与T细胞表面的免疫抑制性受体PD-1结合后，可诱导T细胞凋亡、失能、耗竭，进而抑制肿瘤抗原特异性T细胞的激活、增殖和抗肿瘤功能，实现肿瘤免疫逃逸。PD-1/PD-L1阻断型抗体可以解除PD-L1的免疫抑制作用，增强体内免疫细胞T细胞对肿瘤细胞的识别和杀伤，从而达到杀灭肿瘤的作用。目前，全球范围内已上市多个靶向PD-1/PD-L1的抗体类药物，在临床上对黑色素瘤、肺癌、肾癌、霍奇金淋巴瘤、头颈鳞癌和尿路上皮癌等多种肿瘤显示出不错的治疗效果。目前PD-1/PD-L1治疗性抗体虽然取得了一定的临床效果，但临床上使用有效率偏低，对于绝大多数癌种来说，如果不加选择地单独使用，有效率平均只有10％-20％。因此需要开发更加有效的抗体类药物分子以满足临床需求。
在肿瘤微环境中，癌细胞会通过将PD-L1表达水平上调，躲避人体免疫系统的识别和攻击。CD47蛋白是癌细胞上过度表达的免疫调节分子，主要通过与抑制性受体信号调节蛋白α(SIRPα)作用而抑制巨噬细胞的吞噬作用，并介导多种恶性肿瘤的免疫逃逸。靶向PD-L1/CD47的双特异抗体，可以同时阻断CD47和PD-L1两个通路，有望获得更强的抗肿瘤活性，从而解决临床上PD-L1抗体单独使用有效率偏低的问题。目前临床上尝试将抗PD-L1抗体和抗CD47抗体联用，期望将二者的免疫调节作用叠加，获得更好的治疗效果。而更多的研究者尝试通过构建靶向PD-L1/CD47的双特异抗体，同时阻断CD47和PD-L1两个通路，以期获得更强的抗肿瘤活性，从而解决临床上PD-L1抗体单独使用有效率偏低的问题。
尽管目前PD-L1/CD47双特异性抗体在肿瘤治疗上取得了一定的进展，一定程度上克服了单独使用抗PD-L1和抗CD47的缺陷，但仍存在以下缺陷：1.抗CD47一侧的分子选择上，主要选择SIRPα蛋白作为结合相，而非抗体分子。这种类抗体重组蛋白在稳定性、质量、以及PK/PD特点上都与单抗有较大差距。2.抗体结构上A、B两条链序列结构上的差异给表达、纯化工艺以及质量控制带来较大的难度，轻重链错配率高，正确装配的双特异性抗体分子纯化困难、回收率低。3.PD-L1/CD47双特异性抗体仍存在结合红细胞产生溶血、凝集的可能，需进一步降低对红细胞的结合能力。
由此，抗CD47人源化抗体和PD-L1/CD47双特异性抗体有待进一步研究。
发明内容
为解决上述问题，本发明提供一种抗CD47人源化抗体和PD-L1/CD47 双特异性抗体，其中，抗CD47人源化抗体能结合人CD47，阻断CD47与SIRPα的结合，并且，该抗体为抗CD47的中低亲和力抗体，同时，该抗体仅特异性结合肿瘤细胞表面CD47，而与人红细胞表面CD47不结合，从而解决了抗体结合红细胞产生的溶血和凝集等问题；其中，该双特异性抗体包括两条重链和两条轻链，其中所述两条重链的可变区是异源的，所述两条轻链的可变区序列相同，具体地，针对CD47和PD-L1的双特异性抗体，其中的两条重链的可变区分别衍生自抗CD47单克隆抗体的重链可变区和抗PD-L1单克隆抗体的重链可变区，两条序列相同的轻链是基于抗CD47单克隆抗体轻链和抗PD-L1单克隆抗体轻链序列相似性优化设计产生的。本公开提供的双特异性抗体，具有序列相同的轻链因而能在一定程度上有效解决了双特异性抗体装配过程中轻重链错配的技术问题。
抗CD47抗体
本发明实施例制备了特异性结合人CD47鼠源抗体，在鼠源抗体序列分析的基础上，选择近似的人抗体种系模板制备了人源化抗人CD47单抗或其片段。提供一种具有阻断SIRPα与细胞表面CD47结合的活性以及肿瘤抑制活性的人源化抗人CD47单抗或其片段。进而，在人源化抗人CD47单抗的基础上通过对CDRs区和FR区序列的点突变改造，调整对人CD47的亲和力，并进一步通过轻链突变差异化抗体与肿瘤细胞表面CD47和人红细胞表面CD47的结合活性，获得一系列不具有红细胞结合活性的抗CD47抗体。进一步，选择使人源化抗人CD47单抗在保持CD47特异性结合能力的情况下，容忍轻链CDRs的多个氨基酸位点突变，从而为共轻链双特异性抗体的研发提供新的平台。具体而言：
根据本发明的一个方面，本发明提供一种人源化抗人CD47单抗或其片段。根据本发明的实施例，人源化抗人CD47单克隆抗体或其片段，其特征在于，该人源化抗人CD47单抗或其片段包括：重链CDR1，包括X1YX2MX3，其中X1选自N、S，X2选自V、A，X3选自H、S；重链CDR2，包括YINPX4NX5X6IKYNEKFX7G，其中X4选自Y、G，X5选自D、E，X6选自G、A，X7选自T、Q；重链CDR3，包括EGDFYANYGRLGFX8Y，其中X8选自A、D；并且，轻链CDR1，包括RASQDIX9NYLN，其中X9选自S、T；轻链CDR2，包括YTSRLX10S，其中X10选自H、Q、S；轻链CDR3，包括QQGX11X12X13PX14T，其中X11选自D、A，X12选自T、G，X13选自F、R、Y、K、S、E、N，X14选自Y、R。
根据本发明的实施例，其种系模板选择IGKV1-33*01|IGKJ4*02和IGHV1-3*01|IGHJ1*01。
根据本发明的实施例，该人源化抗人CD47单抗或其片段的CDRs和/或FR区包括点突变，所述点突变有助于减少或消除对红细胞的结合活性，并至少部分地保留人源化抗人CD47单抗或其片段对肿瘤细胞的结合能力。
根据本发明的实施例，所述重链CDR1具有SEQ ID NO:76、77、78或87所示的序列；所述重链CDR2具有SEQ ID NO:79、80、81、82、83或84所示的序列；所述重链CDR3具有SEQ ID NO:85或86所示的序列。
根据本发明的实施例，所述轻链CDR1具有SEQ ID NO:46或47所示的序列；所述轻链CDR2具有SEQ ID NO:50、51或52所示的序列；所述轻链CDR3具有SEQ ID NO:56、57、58、59、60、61、62、63、64、65或68所示的序列。
根据本发明的实施例，人源化抗人CD47单抗或其片段所述的人源化抗人CD47单抗或其片段具有SEQ ID NO:8、11、12、13、14、15、16、17、18、19、20、21、22或23所示重链可变区，SEQ区ID NO:5所示轻链可变区。
根据本发明的实施例，该人源化抗人CD47单抗或其片段所述单克隆抗体为衍生化抗体，所述衍生化抗体包括在初始单克隆抗体的基础上进行CDRs移植、亲和力成熟、点突变改造和化学修饰获得的抗体，其中，所述化学修饰包括糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、蛋白酶切割、与细胞配体或效应分子连接、活性反应基团保护和/或封闭。
进一步，本发明任一所述人源化抗人CD47单抗或其片段，其特征在于：所述人源化抗人CD47单抗或其片段部分包括Fab、Fab’、F(ab’)2、Fv、scFv或dAb。
进一步地，根据本发明另一方面，本发明提供一种人源化抗人CD47单抗或其片段，其是在鼠源抗人CD47单抗的基础上，选择IGKV1-33*01|IGKJ4*02和IGHV1-3*01|IGHJ1*01为种系模板进行人源化得到的。
根据本发明的实施例，该人源化抗人CD47单抗或其片段的重链可变区具有SEQ ID NO:8所示的序列，所述重链可变区具有CDRs序列(HCDR1、HCDR2、HCDR3)，轻链可变区具有SEQ ID NO:5所示的序列，所述轻链可变区具有CDRs序列(LCDR1、LCDR2、LCDR3)。
根据本发明的实施例，该人源化抗人CD47单抗或其片段的重链可变区如SEQ ID NO:8所示，轻链可变区如SEQ ID NO:5所示。
进一步地，根据本发明的实施例，该人源化抗人CD47单抗或其片段具有：
重链CDR1，包括X 1YX 2MX 3，其中X 1选自N、S，X 2选自V、A，X 3选自H、S；
重链CDR2，包括YINPX 4NX 5X 6IKYNEKFX 7G，其中X 4选自Y、G，X 5选自D、E，X 6选自G、A，X 7选自T、Q；
重链CDR3，包括EGDFYANYGRLGFX 8Y，其中X 8选自A、D；
进一步，根据本发明的实施例，该人源化抗人CD47单抗或其片段具有：
轻链CDR1，包括RASQDIX 9NYLN，其中X 9选自S、T；
轻链CDR2，包括YTSRLX 10S，其中X 10选自H、Q、S；
轻链CDR3，包括QQGX 11X 12X 13PX 14T，其中X 11选自D、A，X 12选自T、G，X 13选自F、R、Y、K、S、E、N，X 14选自Y、R。
根据本发明的另一方面，本发明提供了一种多特异性抗体。根据本发明的实施例，所述多特异性抗体至少包括一个针对人CD47的特异性的结合位点，所述针对人CD47的特异性结合位点是由前述人源化抗人CD47单抗或其片段提供的。
根据本发明的实施例，所述多特异性抗体包括针对人CD47不同表位的多个特异性结合位点。
根据本发明的实施例，所述多特异性抗体还包括针对非人CD47靶向的特异性结合位点。
根据本发明的实施例，所述多特异性抗体为双特异性抗体、三特异性抗体或四特异性抗体。
本发明实施例的人源化抗人CD47单抗或其片段至少具有以下有益的技术效果之一：
(1)本发明实施例的人源化抗人CD47单抗或其片段能特异与细胞表面CD47结合，亲和力高，有效阻断癌细胞表面的CD47与SIRPa的相互作用，具有良好的抑瘤效果；
(2)在人源化抗人CD47单抗的基础上通过对CDRs区和FR区序列的点突变改造，调整抗体对人CD47的亲和力，并进一步通过轻链突变差异化抗体与肿瘤细胞表面CD47和人红细胞表面CD47的结合活性，获得一系列对与肿瘤细胞表面CD47结合活性高而对红细胞结合活性低的抗CD47抗体，有效解决了抗CD47抗体在临床上由于红细胞及血小板等结合所带来的抗体浓度低，从而导致临床用药剂量较高的问题，并且，显著降低了抗人CD47抗体的毒副作用，同时，为改善由抗原沉降所导致的体内血药浓度较低的问题提供了新的分子选择。
(3)本发明的人源化抗人CD47单抗能够在保持CD47特异性结合能力的 情况下，容忍轻链可变区多个氨基酸位点突变，从而为共轻链双特异性抗体的研发提供新的平台。
靶向PD-L1和CD47的双特异性抗体或其抗原结合片段
根据本发明的一方面，本发明提供了一种制备双特异性抗体的方法。根据本发明的实施例，该方法包括：
(1)选择轻链种系相同、序列相似的两株单克隆抗体，
(2)构建共表达杂合抗体，检测对第一抗原的结合能力。该杂合抗体的重链和轻链分别包括第一抗体的重链可变区和第二抗体的轻链可变区。
(3)可选的，对所述杂合抗体进行位点突变，构建抗体杂合体群，检测其中每一个抗体对第一抗原的结合能力，从中选定第一重链，该第一重链与第二抗体的轻链形成的杂合抗体对第一抗原的亲和力高，特异性好。
(4)对第二抗体的轻链可变区进行突变构建第二抗体轻链突变体群，，通过与步骤(3)中选定的第一重链共表达制备第一重链/第二抗体突变体杂合抗体，检测对第一抗原的结合能力，选定共同轻链；
(5)根据第二抗体的重链可变区选定第二重链可变区；
(6)将含有步骤(3)选定的第一重链可变区、步骤(4)选定的共同轻链可变区、步骤(5)选定的第二重链可变区的抗体重链和轻链共表达制备双特异性抗体。
根据本发明的实施例，所述步骤(4)中第二抗体轻链突变体可变区的每个氨基酸残基位点至少与第一抗体分子轻链可变区相应位点的氨基酸残基、和/或第二抗体分子轻链可变区相应位点的氨基酸残基相同。
根据本发明的实施例，可选的还包括：(7)检测双特异性抗体对第一抗原和第二抗原的特异性结合能力。
根据本发明的实施例，如果步骤(4)中选定的共同轻链可变区与第二抗体的轻链可变区存在差异，步骤(5)中根据第二抗体的重链可变区选定第二重链可变区包括：构建第二抗体重链可变区突变体群，将其与共同轻链可变区共表达制备第二抗体重链可变区突变体/共同轻链可变区杂合抗体，检测其对第二抗体的结合能力，选定第二重链；
如果步骤(4)中选定的共同轻链与第二抗体的轻链相同，步骤(5)中根据第二抗体的重链可变区选定第二重链可变区即为将第二抗体的重链作为第二重链。
根据本发明的另一方面，本发明提供一种双特异性抗体或其抗原结合片段。根据本发明的实施例，该双特异性抗体或其抗原结合片段包括两条重链和两条轻链，其中，第一重链可变区源自抗CD47单克隆抗体的重链可变区、其能够 与轻链可变区形成CD47结合位点，第二重链可变区源自抗PD-L1单克隆抗体的重链可变区、其能够与轻链可变区形成PD-L1结合位点；其中，所述两条轻链的轻链可变区的序列相同，并且，选自抗CD47单克隆抗体的轻链、抗PD-L1单克隆抗体的轻链、序列结构上介于抗CD47单克隆抗体轻链、抗PD-L1单克隆抗体轻链之间的共同轻链，其中，所述抗CD47单克隆抗体为权利要求1-6任一所述的人源化抗人CD47单抗或其片段。
根据本发明的实施例，所述第一抗体分子的轻链可变区与第二抗体分子的轻链可变区属于相同的抗体轻链种系，具有相同或高度相似的框架区(FR)。
根据本发明的实施例，所述共同轻链可变区具有第一抗体分子轻链可变区和第二抗体分子轻链可变区之间共同的氨基酸残基，并且在第一抗体分子轻链可变区和第二抗体分子轻链可变区之间存在差异的氨基酸位点，所述共同轻链与第一抗体分子轻链相同或与第二抗体分子轻链相同。
根据本发明的实施例，所述双特异性抗体或其抗原结合片段中的第一重链可变区是在第一抗体分子的重链可变区基础上进行点突变获得的，所述第一重链可变区至少部分的保留了第一抗体分子重链可变区与轻链可变区形成第一抗原结合位点的能力。
根据本发明的另一方面，本发明还提供一种靶向PD-L1和CD47的双特异性抗体或其抗原结合片段。根据本发明的实施例，该双特异性抗体或其抗原结合片段包括两条重链和两条轻链，其中，第一重链可变区源自抗CD47单克隆抗体的重链可变区、其能够与轻链可变区形成CD47结合位点，第二重链可变区源自抗PD-L1单克隆抗体的重链可变区、其能够与轻链可变区形成PD-L1结合位点；其中，所述两条轻链的轻链可变区的序列相同，并且，所述轻链可变区是基于抗CD47单克隆抗体的轻链和抗PD-L1单克隆抗体的轻链得到的，且所述轻链可变区与所述第一重链可变区相匹配形成第一可变区结合位点并与CD47特意性结合，所述轻链可变区与所述第二重链可变区相匹配形成第二可变区结合位点并与PD-L1特意性结合，其中，所述抗CD47单克隆抗体为前述的人源化抗人CD47单抗或其片段。
根据本发明的实施例，第一可变区结合位点并与CD47特意性结合，其亲和力与原始的CD47单抗的可变区与与CD47的亲和力相近，并且，第一可变区结合位点并与PD-L1特意性结合，其亲和力与原始的PD-L1单抗的可变区与与PD-L1的亲和力相近。
根据本发明的实施例，所述抗PD-L1单克隆抗体包括SEQ ID NO:3所示重 链可变区和SEQ ID NO:1所示轻链可变区。
根据本发明的实施例，第一重链包括选自SEQ ID NO:8、11、12、13、14、15、16、17、18、19、20、21、22、23的重链可变区。
根据本发明的实施例，所述轻链可变区具有SEQ ID NO:1、24、25、26、27、28、29、30、31、32、33或34所示的序列。
根据本发明的实施例，所述抗体或其抗原结合片段包括但不限于完全抗体、F(ab')2。
根据本发明的实施例，所述完全抗体为IgG1或IgG4型。
根据本发明的实施例，所述两条重链的Fc段包含“knobs-into-holes”突变；
根据本发明的实施例，所述第一重链的Fc段包含H315R突变、另一条所述第二重链的Fc段包括K319E突变，所述Fc段氨基酸突变位点使用Kabat的Eu编号系统。。
根据本发明的又一方面，本发明提供了一种多核苷酸。根据本发明的实施例，该多核苷酸编码前述的人源化抗人CD47单抗或其片段，或前述的多特异性抗体，或前述的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段。
根据本发明的又一方面，本发明提供了一种载体。根据本发明的实施例，该载体包括前述的多核苷酸。
根据本发明的又一方面，本发明提供了一种宿主细胞。根据本发明的实施例，该宿主细胞包括前述的多核苷酸或前述的载体。
根据本发明的又一方面，本发明提供了一种制备抗体或其抗原结合片段的方法。根据本发明的实施例，该方法包括以下步骤：(1)在适合表达双特异性抗体或其抗原结合片段的条件下培养权利要求21所述宿主细胞；(2)从细胞培养物中分离纯化双特异性抗体或其片段。
与现有技术相比，本发明实施例的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段的技术方案至少具有以下优点之一：
第一，本发明实施例制备的双特异性抗体，具有天然抗体的双结合臂，每个结合臂均具有完整的Fab结构，避免了在抗体分子中引入受体链(例如CD47受体SIRPα)等非抗体组件而产生的结构不稳定、功能相互干扰等缺点。进而，通过本发明实施例在共同轻链可变区、重链可变区进行突变改造的基础上，实现了对两条结合臂结合能力的调控，例如制备出对第一抗原/抗原表位具有高度结合能力、并且对第二抗原/抗原表位具有适度结合能力的双特异性抗体。
第二，根据现有技术中抗原结合位点中6个CDRs对抗原结合活性影响程 度的差异，即重链三个CDRs的作用大于轻链三个CDRs、CDR3的作用大于CDR2和CDR1。结合抗PD-L1单克隆抗体hz182和抗CD47单克隆抗体hz140轻链序列结构的相似性，发明人设计了具有相同轻链的抗人PD-L1/CD47双特异性抗体。共同轻链的设计不仅简化了重组表达方法，而且彻底消除了双特异性抗体重组表达过程中轻重链错配的难题，显著简化了生产工艺、提高了产品合格率和均一性。此外，还可在Fc段引入“knobs-into-holes”突变，增强双特异性抗体重链Fc段配对的准确性，避免同源重链二聚化。
第三，在双特异性抗体的设计制备过程中，通过对抗CD47抗体的重链进行有限的氨基酸突变，使得重组抗体中抗CD47的结合臂中重链可变区和轻链可变区均与抗CD47初始单克隆抗体存在结构上微小的差异。通过在上述抗CD47的抗原结合位点上引入序列结构的微小差异使所述双特异性抗体具有适度的抗CD47活性，既保持了双特异性抗体对肿瘤细胞表面CD47的结合活性、增强了抗PD-L1活性的肿瘤抑制作用；又显著降低了对红细胞表面CD47的结合，不仅减少或消除了溶血、红细胞凝集等不期望的后果，而且还能减少全身给药的用量。
抗体的应用和用途
根据本发明的一个方面，本发明提供了一种组合物。根据本发明的实施例，该组合物包含前述的人源化抗人CD47单抗或其片段、前述的多特异性抗体、前述的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段、前述多核苷酸、前述载体和前述宿主细胞中的至少一种。
根据本发明的又一个方面，本发明提供了前述的人源化抗人CD47单抗或其片段、前述的多特异性抗体、前述的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段、前述多核苷酸、和前述组合物在制备治疗肿瘤和/或提高机体免疫应答药物中的用途。
根据本发明的实施例，所述肿瘤包括黑色素瘤、肺癌、肾癌、霍奇金淋巴瘤、头颈鳞癌、尿路上皮癌、急慢性髓系白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤、膀胱癌、卵巢癌、乳腺癌、直肠癌、前列腺癌、肾癌和多发性骨髓瘤。
根据本发明的又一个方面，本发明提供了治疗患有肿瘤的对象的方法。根据本发明的实施例，所述方法包括向所述对象施用治疗有效量的组合物，所述组合物为前述的组合物。
为更好理解本发明，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“PD-L1”，即PD-L1(programmed death ligand 1)全称程序性死亡受体配体1，也称为表面抗原分化簇274(cluster of differentiation 274，CD274)或B7同源体(B7 homolog 1，B7-H1)，由CD274基因编码，是PD-1(programmed cell death 1,程序性死亡受体1)的配体。PD-L1是大小为40kDa的第一型跨膜蛋白，表达在T细胞、B细胞等免疫细胞以及肿瘤细胞上，正常情形下免疫系统会对聚集在淋巴结或脾脏的外来抗原产生反应，促发具抗原特异性的细胞毒杀性T细胞(CD8+Tcell增生)。当肿瘤细胞膜上的PD-L1与T细胞等免疫细胞上的PD-1结合后，肿瘤细胞发出抑制性信号，减低淋巴结CD8+T细胞的增殖，进而导致T细胞不能识别肿瘤细胞和对肿瘤细胞产生杀伤作用，机体的免疫功能受到抑制。
术语“CD47”，又称整合素相关蛋白，分子量约50kDa，在正常细胞和肿瘤细胞中均表达。CD47可与巨噬细胞上蛋白SIRPα结合，提供一种“别吃我”信号，从而抑制巨噬细胞吞噬功能，导致肿瘤的免疫逃逸。在肿瘤细胞、衰老的红细胞和血小板，阻断CD47-SIRPα的相互作用会引发吞噬作用。一方面，根据该作用机制，近些年来CD47在肿瘤免疫治疗中逐渐升温，是肿瘤免疫治疗的一个潜在有效和广泛应用的靶点，许多抑制剂已经被开发出来，可以特异性地阻断CD47-SIRPα信号通路，以增强对肿瘤的免疫应答。然而另一方面，阻断CD47-SIRPα相互作用的机制导致红细胞减少和血小板减少是成药性的最大挑战。
术语“特异性”是指在蛋白和/或其他生物异质群体中确定是否存在所述蛋白，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著的量与样本中存在的其它蛋白结合。
本文中的术语“抗体”意在包括全长抗体及其任何抗原结合片段(即，抗原结合部分)或单链。全长抗体是包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接。各重链由重链可变区(简称VH)和重链恒定区构成。重链恒定区由三个结构域构成，即CH1、CH2和CH3。各轻链由轻链可变区(简称VL)和轻链恒定区构成。轻链恒定区由一个结构域CL构成。VH和VL区还可以划分为称作互补决定区(CDR)的高变区，其由较为保守的框架区(FR)区分隔开。各VH和VL由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括多种免疫系统细胞(例如，效应细胞)和传统补体系统的第一 组分(C1q)。
术语“单克隆抗体”或“单抗”或“单克隆抗体组成”是指单一分子组成的抗体分子制品。单克隆抗体组成呈现出对于特定表位的单一结合特异性和亲和力。
本文中的术语，抗体的“抗原结合片段”(或简称为抗体部分)，是指抗体的保持有特异结合抗原能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施。包含在抗体的“抗原结合部分”中的结合片段的例子包括(i)Fab片段，由VL、VH、CL和CH1构成的单价片段；(ii)F(ab′)2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1构成的Fd片段；(iv)由抗体单臂VL和VH构成的Fv片段；(v)由VH构成的dAb片段(Ward et al.，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域VL和VH由不同的基因编码，它们可以通过重组法经由使两者成为单蛋白链的合成接头而连接，其中VL和VH区配对形成单价分子(称为单链Fc(scFv)；参见例如Bird et al.，(1988)Science 242：423-426；and Huston et al.，(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883)。这些单链抗体也意在包括在术语涵义中。这些抗体片段可以通过本领域技术人员已知的常用技术而得到，且片段可以通过与完整抗体相同的方式进行功能筛选。
本发明的抗原结合片段包括能够特异性结合抗原分子的那些。抗体结合片段的实例包括例如但不限于Fab、Fab'、F(ab')2、Fv片段、单链Fv(scFv)片段和单结构域片段。
Fab片段含有轻链的恒定结构域和重链的第一恒定结构域(CH1)。Fab'片段与Fab片段的不同之处在于在重链CH1结构域的羧基末端处的少数残基的添加，包括来自抗体铰链区的一个或多个半胱氨酸。通过切割在F(ab')2胃蛋白酶消化产物的铰链半胱氨酸处的二硫键产生Fab'片段。抗体片段的另外化学偶联是本领域普通技术人员已知的。Fab和F(ab')2片段缺乏完整抗体的片段可结晶(Fc)区，从动物的循环中更快速地清除，并且可能具有比完整抗体更少的非特异性组织结合(参见例如，Wahl等人，1983，J.Nucl.Med.24:316)。
如本领域通常理解的，“Fc”区是不包含抗原特异性结合区的抗体的片段可结晶恒定区。在IgG、IgA和IgD抗体同种型中，Fc区由两个相同的蛋白质片段组成，衍生自抗体的两条重链的第二和第三恒定结构域(分别为CH2和CH3结构域)。IgM和IgE Fc区在每条多肽链中含有三个重链恒定结构域(CH2、CH3和CH4结构域)。
“人源抗体”包括具有人免疫球蛋白的氨基酸序列的抗体，并且包括从人 免疫球蛋白文库或动物中分离的抗体，所述动物对于一种或多种人免疫球蛋白是转基因的，并且不表达内源免疫球蛋白。人抗体可以通过本领域已知的各种方法制备，所述方法包括使用衍生自人免疫球蛋白序列的抗体文库的噬菌体展示方法。参见美国专利号4,444,887和4,716,111；以及PCT公开WO 98/46645；WO 98/50433；WO 98/24893；WO 98/16654；WO 96/34096；WO96/33735；和WO 91/10741。还可以使用不能表达功能性内源免疫球蛋白，但可以表达人免疫球蛋白基因的转基因小鼠来产生人抗体。参见例如，PCT公开WO 98/24893；WO 92/01047；WO 96/34096；WO 96/33735；美国专利号5,413,923；5,625,126；5,633,425；5,569,825；5,661,016；5,545,806；5,814,318；5,885,793；5,916,771；和5,939,598。另外，使用与上述类似的技术，公司例如LakePharma，Inc.(Belmont，CA)或Creative BioLabs(Shirley，NY)可以从事于提供针对所选抗原的人抗体。可以使用被称为“引导选择”的技术生成识别所选表位的全人抗体。在该方法中，选择的非人单克隆抗体，例如小鼠抗体，用于引导识别相同表位的完全人抗体的选择(参见，Jespers等人，1988，Biotechnology 12:899-903)。
术语“重链”，重链(heavy chain，H链)大小约为轻链的2倍，含450～550个氨基酸残基，分子量约为55或75kD。每条H链含有4～5个链内二硫键所组成的环肽。不同的H链由于氨基酸组成的排列顺序、二硫键的数目和位置、含的种类和数量不同，其抗原性也不相同，根据H链抗原性的差异可将其分为5类：μ链、γ链、α链、δ链和ε链，不同H链与L链(κ或λ链)组成完整免疫球蛋白的分子分别称之为IgM、IgG、IgA、IgD和IgE。γ、α和δ链上含有4个肽，μ和ε链含有5个环肽。
术语“轻链”，轻链(light chain，L)指在免疫球蛋白单体分子中相对于重链而论，在分子量上较小的多肽链。每条轻链近氨基末端(N端)1/2区域内的氨基酸组成序列多变处为轻链可变区(VL)，是Ig分子与抗原结合部位的一个组成部分。其余1/2区域内的氨基酸组成及排列顺序相对稳定处为轻链恒定区(CL)。由于轻链恒定区内氨基酸序列存在某些差异，故轻链有k和λ两型。
术语“种系”，又称胚系(germline)，抗体形成细胞具有编码Ig分子的全部基因(即有限数量的C基因和未知数量的V基因，它是通过长期进化形成并通过生殖细胞从亲代传给子代，人免疫球蛋白的重链基因由V-D-J-C基因片段编码而成；轻链基因由V-J-C基因片段编码而成，基因片段的个数不等。人重链基因定位于第14号染色体长臂，跨度约1,100kb，由V、D、J和C四种基因片段组成，包含约95个Va基因片段(分VHl～VH7七个家族，其中功能性基因片段65个、27个D基因片段、6个JH基因片段和9个CH基因片段。Va基 因片段位于上游，D基因片段位于VH和JH基因簇之间，JH基因位于DH下游，与下游C基因区相隔7Kb左右，CH基因成簇(cluster)排列，跨度约200kb，P和S基因紧位于JH基因片段下游，C8下游依次是Cγ、Cα和Cε。人轻链基因分为λ和x基因，分别定位于第22号染色体长臂和第2号染色体短臂。功能性VK基因片段约40个，Vc基因片段后是5个功能性J和1个Cκ；Vλ基因片段约30个，4个Jλ基因片段和4个Cλ基因片段。
术语“EC50”，又叫半最大效应浓度，是指引起50％最大效应的抗体浓度。
术语“双特异性抗体”(bispecific antibodies)，一种可与相同或不同抗原上的不同表位结合的抗体结构。因此，双特异性抗体能够桥连两种不同的分子，起到将效应分子、效应细胞、病毒和药物载体系统招募至靶标结构的作用。双特异性抗体这种可同时识别两种不同分子(受体和/或配体)的特点，提高了抗体的选择性和功能性亲和力。
术语“错配”(mispair)，类IgG结构的双特异性抗体通常采用单细胞进行表达，但抗体由2条轻链(L)和2条重链(H)构成，用单细胞系共表达的体系中理论上存在轻重链随机错配的问题，可能出现10种HHLL的组合，而实际上具有应用意义的目标异源抗体组合仅占12.5％。从含近10种混合物的复杂体系中纯化出足量的目标抗体极富挑战性，因此带来的问题是产品的均一性和产率较低、后续纯化工艺复杂。上市产品Catumaxomab(抗体类型为Triomab)的生产策略在此基础上进行了改进，Triomab采用大鼠杂交瘤和小鼠杂交瘤进行体细胞融合(somatic fusion)，其种属限制性H-L链配对(Species restricted H-Lchain pairing)的策略可减少随机轻重链错配的比例(＜10％)。根据不同种属和不同亚类(subclass)的抗体Fc段对蛋白A介质的结合能力不同，通过一步蛋白A亲和层析纯化即可分离出同时具有大鼠Fc段和小鼠Fc段的目标抗体，因此简化了纯化步骤。但是，基于Triomab技术构建的双特异性抗体为鼠源抗体，具有很强的免疫原性，因此其临床应用范围受到限制。
需要说明的是，根据本发明实施例的确定DNA样品的序列信息的方法是本申请的发明人经过艰苦的创造性劳动和优化工作才完成的。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1：Fortebio测定Hz140与CD47-his的亲和力结果示意图；
图2：FACS分析hz140与肿瘤细胞U937的结合活性结果示意图；
图3：FACS分析hz140与肿瘤细胞CCRF的结合活性结果示意图；
图4：FACS分析hz140与红细胞的结合活性结果示意图；
图5：FACS分析hz140对SIRPa与CCRF细胞表面CD47阻断活性结果示意图；
图6：FACS分析hz140对SIRPa与U937细胞表面CD47阻断活性结果示意图；
图7：hz140对CCRF-SB白血病模型小鼠生存期观测结果结果示意图；
图8：抗CD47抗体对人源异种移植淋巴瘤Raji模型的体内药效观察结果示意图；
图9：2MW1531对免疫缺陷小鼠尾静脉异种移植Raji血液肿瘤模型抗肿瘤药效检测信号强度定量结果示意图；
图10：hz140轻链和hz182轻链氨基酸序列比对结果示意图；
图11：2MW1531与MC38-hPD-L1/hCD47细胞结合活性分析结果示意图；
图12：2MW1531与A431细胞结合活性分析结果示意图；
图13：2MW1531与人红细胞结合活性分析结果示意图；
图14：抗人CD47/PD-L1双特异抗体体外红细胞凝集试验结果示意图；
图15：2MW1531对hPD-L1/hCD47/hSIRPα转基因小鼠皮下同种移植MC38-hPDL1鼠结肠癌肿瘤模型的抗肿瘤药效实验结果示意图(肿瘤体积)；
图16：2MW1531对免疫缺陷小鼠尾静脉异种移植Raji血液肿瘤模型抗肿瘤药效检测活体成像图。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Sigma公司。
实施例1：抗CD47高亲和力抗体140及对照抗体的制备
CD47-mFc免疫Balb/c小鼠进行杂交瘤制备。并分别于初次免疫后34天或57天进行血清抗体滴度检测。挑选血清滴度达到融合实验要求的小鼠(血清抗体效价：1:102400)脾细胞与骨髓瘤细胞FO以适当的比例(5:1)，在融合剂的作用下进行融合，制备杂交瘤单克隆细胞。利用选择性培养基R1640-HAT对杂交瘤细胞进行培养，于第10-14天采用ELISA的方法对651个杂交瘤上清样品进行检测，包括细胞结合活性(FACS)、CD47/SIPR□阻断、以及与重组cyno-CD47-mFc的交叉反应情况，筛选出克隆140。调取上述克隆轻重链可变区基因，并将其轻重链可变区基因克隆入含有人Cκ和IgG4基因阅读框的重组表达载体，在HEK293细胞上进行瞬时表达，纯化后获得蛋白纯品进行分析，克隆140鼠源抗体轻、重链可变区氨基酸序列分别为SEQ ID NO:43和SEQ ID NO:44。
SEQ ID NO.43:鼠源140轻链可变区氨基酸序列
Figure PCTCN2021130320-appb-000001
SEQ ID NO.44:鼠源140重链可变区氨基酸序列
Figure PCTCN2021130320-appb-000002
制备如下抗体作为参照抗体，均采用IgG4的形式。对照抗体的重组表达策略采用常规分子生物学方法实现，利用HEK293瞬时表达完成表达和纯化。Forty Seven公司临床在研抗体hu5F9(US2015/0183874A1，hu5F9V2,轻重链可变区序列：SEQ ID NO.35和SEQ ID NO.36)，I-Mab公司的抗CD47抗体1F8(CN 110582515 A，轻重链可变区SEQ IDNO.37和SEQ ID NO.38)。
SEQ ID NO.35:hu5F9轻链可变区氨基酸序列
Figure PCTCN2021130320-appb-000003
Figure PCTCN2021130320-appb-000004
SEQ ID NO.36:hu5F9重链可变区氨基酸序列
Figure PCTCN2021130320-appb-000005
SEQ ID NO.37:1F8轻链可变区氨基酸序列
Figure PCTCN2021130320-appb-000006
SEQ ID NO.38:1F8重链可变区氨基酸序列
Figure PCTCN2021130320-appb-000007
实施例2：抗人CD47抗体140的人源化改造及评价
2.1鼠源抗体140的人源化
对鼠源抗体140进行人源化设计，选择IGKV1-33*01|IGKJ4*02和IGHV1-3*01|IGHJ1*01为germline模板。人源化后轻重链序列见SEQ ID NO.8和SEQ ID NO.5。
SEQ ID NO.8 140人源化后重链可变区氨基酸序列
Figure PCTCN2021130320-appb-000008
SEQ ID NO.5 140人源化后轻链可变区氨基酸序列
Figure PCTCN2021130320-appb-000009
2.2细胞表面结合和阻断活性评价
分别利用Fortebio和FACS对人源化140进行了评价。Fortebio结果如图1和表1所示。将不同浓度的人源化140(hz140)上样至固定有CD47-His的表面，所述hz140的浓度分别为75nm、60nm、30nm、15nm，经测定kon(1/Ms)＝2.47E+05，kdis(1/s)＝3.70E-04、KD(M)＝1.50E-09。提示人源化后的hz140保持了较好的亲和力。
表1：Fortebio测定Hz140与CD47-his的亲和常数
Figure PCTCN2021130320-appb-000010
利用FACS对hz140与U937、CCRF和人RBC细胞表面的CD47抗原的结合活性进行评价。结果如图2-图4所示，在三种细胞上hz140与对照抗体hu5F9的结合曲线相似，表明hz140在三种细胞表面的结合活性均与对照抗体hu5F9基本相当。SIRPa-Fc(AAH26692.1，FITC labeled rhSIRPa-Fc)与U937和CCRF细胞表面的CD47结合阻断实验结果如图5和图6所示，在CD47丰度较高的U937细胞上，hz140的阻断活性与hu5F9基本相当；在CD47丰度相对较低的CCRF细胞上，hz140的阻断活性明显强于hu5F9。
2.3 Hz140对人急性B淋巴细胞的白血病模型CCRF-SB的抗肿瘤药效
Balb/c裸鼠，6-8周，腹腔注射环磷酰胺2mg/只/200μl进行免疫抑制，48小时后尾静脉接种新鲜处理的CCRF-SB细胞6.25×106。接种5天后(D5)，测量小鼠体重，按照体重数据将小鼠随机分配到各实验组中，每组8只。分组当天开始分别给予不同剂量受试抗体(i.v.，每周2次，共6次)，并观察小鼠的生存期。结果如图7所示，与hIgG阴性对照相比，阳性对照抗体hu5F9能够延长模型小鼠的生存期，但hu5F9的高剂量组(10mg/kg)和低剂量组(3.3mg/kg)之间无明显差异。hz140不仅与阴性对照相比显著延长了模型小鼠的生存期，而且效果优于相同剂量的阳性对照抗体hu5F9。另外，hz140高剂量组的效果优于hz140低剂量组，提示hz140对延长模型小鼠生存期呈现出剂量依赖性。
上述结果表明本发明制备的hz140在人急性B淋巴细胞白血病动物模型中治疗效果优于同剂量的hu5F9，且呈现剂量依赖性、具有更高的可靠性。
实施例3.Hz140的亲和力改造
在人源化hz140抗体轻重链可变区序列(SEQ ID NO.8)的基础上，对重链CDRs序列进行突变设计，通过降低抗体与CD47结合的Koff值以期降低其对RBC及PLT的影响。构建了多个的人源化hz140的重链突变体，对人源化hz140的重链进行氨基酸定点突变，将突变的重链与人源化hz140的轻链配对表达，测定亲和力，比较结果如下表2所示。并进一步在hm10的基础上进行 突变，获得140Hm13最终的序列如SEQ ID NO.23所示。
表2.hz140-Hm/hz140L重链突变体序列信息及其与CD47胞外区重组抗原的亲和力
Figure PCTCN2021130320-appb-000011
SEQ ID NO.23：人源化140抗体的重链突变体140Hm13
Figure PCTCN2021130320-appb-000012
实施例4红细胞结合活性降低突变体筛选
以140Hm13为重链，分别与h140L(SEQ ID NO.5)和另一异源抗体的相似轻链序列hz182L(SEQ ID NO.1)进行突变体设计和组合，评估其突变体与肿瘤细胞表面CD47和红细胞表面CD47的结合差异。对hz140的轻链可变区序列进行突变设计，突变体设计如下表3所示；对hz182的轻链(h182L)进行突变设计，突变体设计如下表4所示。通过FACS评估不同突变体与肿瘤细胞 (1μg/mL)和红细胞(150μg/mL)的结合情况，寻找具有结合差异的突变位点。
SEQ ID NO.1：人源化182抗体的轻链序列hz182L
Figure PCTCN2021130320-appb-000013
将hz140重链突变体h140Hm13，分别与h140轻链系列突变体(h140Lm1～h140Lm18)、h182轻链系列突变体(hz182-Lm1～hz182-Lm11)组合共表达制备重组抗体，发现了一系列与红细胞无特异性结合的抗CD47特异性抗体。根据表3、表4中h140L和h182L的突变体结果，发现h140Lmut8-mut10的红细胞结合能力明显降低，同时肿瘤的结合活性虽然降低，但仍然保持一定的结合活性。尤其是mut8和mut10，红细胞结合能力完全丧失，但肿瘤细胞结合能力保持，提示位于CDR3区的96位氨基酸从R突变为Y后，是红细胞结合的关键位点。而h182L的反向突变也证实，将h182L突变为h182Lmut8(Y突变为R),其红细胞结合活性恢复。
表3.H140L突变体氨基酸序列
Figure PCTCN2021130320-appb-000014
Figure PCTCN2021130320-appb-000015
表4.H182L突变体氨基酸序列
Figure PCTCN2021130320-appb-000016
实施例5：初始单克隆抗体的制备
1.抗人PD-L1单克隆抗体的制备
抗人PD-L1人源化抗体根据在先申请专利(专利申请号：CN201911419802.5)制备。该人源化抗体的轻链可变区(hz182-L)氨基酸序列见序列SEQ ID NO.1，编码核苷酸序列见序列SEQ ID NO.2；重链可变区(hz182-H)氨基酸序列见序列SEQ ID NO.3，编码核苷酸序列见序列SEQ ID NO.4。
2.抗人CD47单克隆抗体的制备
采用实施例2制备的抗人CD47抗体140，hz140的轻链可变区(hz140-L)氨基酸序列见SEQ ID NO.5，核苷酸序列见SEQ ID NO.6；轻链恒定区氨基酸序列见SEQ ID NO.7；重链可变区(hz140-H)氨基酸序列见SEQ ID NO.8，核苷酸序列见SEQ ID NO.9；重链恒定区氨基酸序列见SEQ ID NO.10。
实施例6：抗CD47抗体重链/抗PD-L1抗体轻链杂合抗体(hz140-H/hz182-L)的制备
通过序列比对，发现实施例2中的hz182抗体的轻链(hz182-L)和hz140抗体的轻链(hz140-L)序列高度相似(同源性96.26％，图10)。图10的序列比对结果显示：hz140轻链和hz182轻链在序列结构上共有8个氨基酸残基的差异，其中CDR1区和FR2区各有1个氨基酸残基的差异，FR3区有2个氨基酸残基的差异，CDR3区有4个氨基酸残基的差异。
试将hz182(hz182-L)的轻链和hz140的重链(hz140-H)进行组合表达，表达上清进行亲和纯化，获得与hz182共轻链的抗人CD47抗体hz140-H/hz182-L。利用Fortebio公司的Octet QKe仪器测定抗体的亲和力，将抗体固定在抗人Fc捕获抗体的传感器表面(AHC Sensor)，与液相中的CD47胞外区重组抗原反应(购自义翘神州，Cat:12283-H08H；工作浓度为100nM)，测定动力学参数。结果显示，hz140-H-/hz182-L与CD47重组蛋白具有良好的结合活性(表5)。
表5.Hz140、hz140-H/hz182-L亲和力检测结果
Figure PCTCN2021130320-appb-000017
实施例7：抗CD47抗体重链突变体/抗PD-L1抗体轻链杂合抗体(hz140-Hm/hz182-L)的制备
对杂合抗体hz140-H/hz182-L的重链进行基因工程改造，并评估对重组抗体对CD47的亲和力。具体的，通过分析抗体可变区各个位点氨基酸种类的分布频率，在可能影响抗体亲和力的氨基酸位点，合理设计突变，并进行不同突变位点的组合；随后通过定点突变PCR将突变引入抗体编码基因相应位点，构建突变体表达质粒。最后将突变后的重链hz140-Hm和轻链hz182-L进行组合表达，表达上清进行亲和纯化。获得的突变体抗体和CD47重组蛋白间的亲和力通过Fortebio进行测定，将抗体固定在抗人Fc捕获抗体的传感器表面(AHC Sensor)，与液相中的CD47胞外区重组抗原反应(工作浓度为100nM)，结合时间和解离时间均为300秒，测定动力学参数。重链hz140-H的突变位点、突变体hz140-Hm与重组CD47的亲和力如表6所示。
实施例8：抗CD47抗体重链突变体13/抗PD-L1抗体轻链突变体杂合抗体(hz140-Hm13/hz182-Lm)的制备
对杂合抗体hz140-Hm13/hz182-L的轻链进行基因工程改造，并评估对重组抗体对CD47的亲和力。所述轻链突变体设计原则如下：保留hz140抗体轻链和hz182抗体轻链之间共有的氨基酸残基，突变存在差异的氨基酸残基，突变的氨基酸残基优先是hz140抗体轻链或hz182抗体轻链该位点的氨基酸，也可以是其他类型氨基酸。随后采用实施例2中的方法检测了这些突变体和CD47间的亲和力。轻链hz182-L突变位点、突变体hz182-Lm与重组CD47的亲和力如表7所示。
表6.hz140-Hm/hz182-L重链突变体序列信息及其与CD47胞外区重组抗原的亲和力
Figure PCTCN2021130320-appb-000018
Figure PCTCN2021130320-appb-000019
表7.hz140-Hm13/hz182-L轻链突变体序列信息及与重组CD47的亲和力
Figure PCTCN2021130320-appb-000020
Figure PCTCN2021130320-appb-000021
实施例9：抗CD47/PD-L1双特异抗体2MW1531-p及其突变体的制备与效果评估
1、抗CD47对照抗体制备
参照专利，合成抗CD47对照抗体hu5F9(公开号：US 9017675B2)和1F8(公开号：CN 110582515 A)的轻、重链可变区的编码基因，将轻、重链可变区编码基因分别克隆至装有人轻链恒定区和人IgG4重链恒定区编码基因的真核表达载体中，获得轻、重链表达质粒，转入大肠杆菌扩增，分离获得大量质粒，利用该质粒，根据转染试剂293fectin的操作说明，将对照抗体的轻、重链质粒转入HEK293细胞中重组表达。细胞转染后5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得对照抗体。本申请中，对照抗体hu5F9的轻链可变区氨基酸序列见SEQ ID NO.35，重链可变区氨基酸序列见SEQ ID NO.36。对照抗体1F8的轻链可变区氨基酸序列见SEQ ID NO.37，重链可变区氨基酸序列见SEQ ID NO.38。
2、抗人CD47/PD-L1双特异抗体2MW1531-p及其突变体的制备
将上述hz140-H突变体的可变区氨基酸序列(SEQ ID NO.23)的编码基因与KIH的A链的恒定区氨基酸序列(SEQ ID NO.39)的编码基因串联克隆到瞬时表达载体中，构建双特异抗体重链hz140-H-A(氨基酸序列：SEQ ID NO.40)的表达载体；将上述hz182-H的可变区氨基酸序列(SEQ ID NO.3)的编码基因(SEQ ID NO.4)与KIH的B恒定区氨基酸序列(SEQ ID NO.41)的编码基因串联克隆到瞬时表达载体中，构建双特异性抗体重链hz182-H-B(氨基酸序列SEQ ID NO.42)的表达载体。然后将两条重链表达载体和两个抗体的共同轻链或其突变体的表达载体(轻链及其突变体可变区氨基酸序列：SEQ ID NO.1,24,25,26,27,28,29,30,31,32,33,34)同时转入真核细胞中表达，并通过亲和纯化获得共轻链“knobs-into-holes”形式的双特异抗体，命名为2MW1531-p及2MW1531-m1～m11。突变体信息如下表所示。2MW1531 hz140-H的Fc突变位点为：H315R，突变后的恒定区氨基酸序列(命名为hz140-H-knobs，A链Fc)见SEQ ID NO.39，hz140-H-A氨基酸序列见SEQ ID NO.40，hz182-H的Fc突变位点为：K319E，突变后的恒定区氨基酸序列(命名为hz182-H-holes，B链Fc)见SEQ ID NO.41；hz182-H-B氨基酸序列见SEQ ID NO.42。轻、重链匹配信息见表8。
表8.双特异抗体2MW1531-p及其突变体轻重链匹配信息表
Figure PCTCN2021130320-appb-000022
Figure PCTCN2021130320-appb-000023
3、CD47/PD-L1双特异抗体亲和力分析
利用Fortebio测定2MW1531-p及其突变体的亲和力。对各个突变体与PD-L1、CD47的亲和力分析如下表9所示。
表9.双特异抗体2MW1531-p及其突变体亲和力测定结果
Figure PCTCN2021130320-appb-000024
Figure PCTCN2021130320-appb-000025
4、CD47/PD-L1双特异抗体2MW1531-p及其突变体与人红细胞和肿瘤细胞结合活性分析
通过FACS对2MW1531-p及突变体与人红细胞和肿瘤细胞(SKOV-3，该肿瘤细胞株体外培养时不表达PD-L1)的结合活性进行了分析。在分析和人红细胞结合的实验中，选取的抗体浓度为1000nM；分析和肿瘤细胞结合的实验中，选取的抗体浓度为100nM。分析结果如下表10所示，2MW1531-p及大部分突变体与人红细胞不结合或弱结合，但对肿瘤细胞呈现良好的结合活性。
表10.双特异抗体2MW1531-p及其突变体与人红细胞和肿瘤细胞结合活性分析结果
Figure PCTCN2021130320-appb-000026
实施例10：抗CD47/PD-L1双特异抗体2MW1531与细胞表面抗原结合活性分析
1.抗人CD47/PD-L1双特异抗体2MW1531与MC38-hPD-L1/hCD47细胞结合活性分析
利用CD47和PD-L1都进行了人源化改造的小鼠黑色素瘤细胞株MC38-hPD-L1/hCD47对2MW1531(m9)的结合活性进行了流式细胞术分析。结果如图11所示，结果显示，对于表达人CD47和人PD-L1的小鼠黑色素瘤细胞株MC38-hPD-L1/hCD47的结合能力：抗人CD47/PD-L1双特异性抗体2MW1531高于对CD47具有高度特异性结合能力的5F9对照单抗、抗PD-L1单抗(轻链/重链可变区氨基酸序列为SEQ ID NO.1/SEQ ID NO.3，恒定区氨基酸序列为SEQ ID NO.7/SEQ ID NO.10)；抗CD47单抗(轻链/重链可变区序列为SEQ ID NO.5/SEQ ID NO.8，恒定区序列为SEQ ID NO.7/SEQ ID NO.10)。图11的结果表明：2MW1531及其抗CD47侧臂单抗和抗PD-L侧臂单抗均与MC38-hPD-L1/hCD47细胞有良好的结合活性，2MW1531的结合信号高于同源的两个单抗，提示双抗对细胞的结合活性具有叠加或协同效应。
2.抗人CD47/PD-L1双特异抗体2MW1531与A431细胞结合活性分析
利用同时表达CD47和PD-L1的人皮肤鳞癌细株A431对2MW1531(m9)的结合活性进行流式细胞术分析，结果如图12所示，实验结果显示，对于同时表达CD47和PD-L1的人皮肤鳞癌细胞株A431的结合能力，抗人CD47/PD-L1双特异性抗体2MW1531高于抗PD-L1单抗、以及对照抗CD47单抗1F8。上述结果表明，2MW1531的结合活性强于同源单特异性抗PD-L1单抗和对照抗CD47单抗1F8。
实施例11：抗人CD47/PD-L1双特异抗体2MW1531与红细胞表面抗原结合活性
取人新鲜抗凝外周血，人红细胞分离试剂盒分离红细胞。分为5×10 5cells/样品/100μL，加入梯度稀释的2MW1531(m7)，加入的抗体终浓度为200nM。冰上孵育2h，冰冷PBS(含0.05％吐温)洗涤细胞2遍；加入FITC标记的抗人Fc二抗(厂家，货号)，冰上孵育1h，冰冷PBS(含0.05％吐温)洗涤细胞2遍，重悬于200μL流式缓冲液中，流式细胞仪检测。检测结果如图13所示。图13的流式细胞仪检测结果显示，2MW1531和阴性对照抗体Isotype对人红细胞的结合无显著差异，抗CD47单抗1F8对人红细胞有结合活性，抗CD47单抗5F9对人红细胞有高度结合能力。
图13的结果表明当前条件下，2MW1531不结合人红细胞，1F8有微弱结合，5F9和人红细胞有很强的结合。
实施例12：抗人CD47/PD-L1双特异抗体2MW1531体外RBC凝集试验
取人、人CD47转基因C57小鼠(hCD47/C57)、食蟹猴新鲜抗凝外周血，红细胞分离试剂盒分离红细胞；计数红细胞并调整细胞密度，加入U型底96孔细胞板至1X10 7细胞/孔/100μL；PBS稀释2MW1531(m11)，800μg/ml起始，4倍稀释7个梯度，等体积(100 μL)加入细胞孔中，轻轻混匀，使细胞终浓度为0.5X10 7细胞/孔/200μL，待测抗体最终工作浓度为400μg/ml起始；将加入样品后的细胞板放置于37℃细胞培养箱孵育4h；肉眼观察凝血现象，判断标准为红细胞沉积于底部且上清清亮为不凝集，上清清亮但红细胞在培养板底部弥散分布为凝集，上清浑浊呈红棕色为溶血。
结果如图14所示，实验结果显示在对人红细胞、CD47人源化的C57小鼠红细胞、猴红细胞的凝集实验结果中，抗CD47单抗5F9和1F8无红细胞凝集现象，2MW1531和NC-IgG4红细胞全部集中于微孔板底部，无网状凝集产生。上述结果表明，2MW1531在当前浓度条件下，未见红细胞聚集或溶血现象。
实施例13：抗人CD47/PD-L1双特异抗体2MW1531体内抗肿瘤活性
1. 2MW1531对hPD-L1/hCD47/hSIRPα转基因小鼠皮下同种移植MC38-hPDL1鼠结肠癌肿瘤模型的抗肿瘤药效检测
将PD-L1和CD47双人源化的鼠结肠癌肿瘤细胞hPDL1/CD47-MC38的接种于雌性hPD-L1/hCD47/hSIRPα转基因小鼠(C57来源的hPD-L1/hCD47/hSIRPα转基因小鼠)右侧前胁肋部皮下，在肿瘤生长至50-100mm3左右时分组并腹腔给予2MW1531(m7)或同型对照IgG，给药剂量为20mg/kg、10mg/kg，每周2次。每次给药的同时测量肿瘤体积及体重，记录荷瘤鼠体重和肿瘤体积的变化与给药时间的关系，结果如图15所示，图15中，结肠癌肿瘤动物模型体内试验结果显示，在给药同等剂量(10mg/kg)的情况下2MW1531对肿瘤体积的抑制作用强于抗CD47单抗，与抗PD-L1单抗相比较效果相当或略强。在加大剂量的情况下(20mg/kg)，2MW1531对肿瘤体积的抑制作用进一步显著提高。上述结果表明，双特异性抗体2MW1531有效地抑制肿瘤的生长，且成剂量依赖性，其活性强于CD47和PD-L1的单抗。
2. 2MW1531对裸小鼠Raji-Luc血液肿瘤模型的抗肿瘤药效检测
为了检测双特异性抗体2MW1531中抗CD47结合臂对肿瘤治疗的作用，进一步采用不表达PD-L1的Raji-Luc细胞进行动物体内。在免疫缺陷小鼠(NCG)尾静脉异种移植Raji-Luc血液肿瘤模型上，对2MW1531(m11)的抗肿瘤药效进行了分析。首先将血液肿瘤细胞Raji-Luc经尾静脉异种移植接种在雌性免疫缺陷小鼠(NCG)体内，随后借助小鼠活体成像系统，通过测定小鼠体内的荧光信号强度来指示血液肿瘤细胞的增殖情况，结果如图16和9所示，图16的活体成像图显示，在不表达PD-L1的Raji肿瘤细胞移植小鼠模型中，抗人CD47/PD-L1双特异性抗体2MW1531对Raji肿瘤细胞生长具有显著抑制作用，对Raji肿瘤细胞增殖的抑制效果与抗CD47单抗5F9相当；图9对放射信号进行定量分析的结果显示，静脉接种Raji肿瘤细胞后利用2MW1531进行腹腔内注射治疗，每周2次，能够显著抑制肿瘤细胞生长，治疗效果与抗CD47单抗5F9相当。上述结果表明，接种在NCG小鼠体内的Raji-Luc细胞可以快速增殖，给予2MW1531治疗后可抑制并清除小鼠体内的肿瘤细胞，提示当肿瘤细胞上不存在PD-L1靶点的情况下，2MW1531可以通过单侧臂阻断CD47信号通路，进而促巨噬细胞吞噬作用，发挥抗肿瘤药效。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims (29)

  1. 一种人源化抗人CD47单克隆抗体或其片段，其特征在于，包括：
    重链CDR1，包括X 1YX 2MX 3，其中X 1选自N、S，X 2选自V、A，X 3选自H、S；
    重链CDR2，包括YINPX 4NX 5X 6IKYNEKFX 7G，其中X 4选自Y、G，X 5选自D、E，X 6选自G、A，X 7选自T、Q；
    重链CDR3，包括EGDFYANYGRLGFX 8Y，其中X 8选自A、D；
    并且，
    轻链CDR1，包括RASQDIX 9NYLN，其中X 9选自S、T；
    轻链CDR2，包括YTSRLX 10S，其中X10选自H、Q、S；
    轻链CDR3，包括QQGX 11X 12X 13PX 14T，其中X 11选自D、A，X 12选自T、G，X 13选自F、R、Y、K、S、E、N，X 14选自Y、R。
  2. 如权利要求1所述的人源化抗人CD47单克隆抗体或其片段，其特征在于，其种系模板选自IGKV1-33*01|IGKJ4*02和IGHV1-3*01|IGHJ1*01。
  3. 如权利要求1或2所述的人源化抗人CD47单克隆抗体或其片段，其特征在于，所述人源化抗人CD47单抗或其片段的CDRs区和/或FR区包括点突变。
  4. 如权利要求1所述的人源化抗人CD47单克隆抗体或其片段，其特征在于，所述重链CDR1具有SEQ ID NO:76、77、78或87所示的序列；
    所述重链CDR2具有SEQ ID NO:79、80、81、82、83或84所示的序列；
    所述重链CDR3具有SEQ ID NO:85或86所示的序列。
  5. 如权利要求1所述的人源化抗人CD47单克隆抗体或其片段，其特征在于，所述轻链CDR1具有SEQ ID NO:46或47所示的序列；
    所述轻链CDR2具有SEQ ID NO:50、51或52所示的序列；
    所述轻链CDR3具有SEQ ID NO:56、57、58、59、60、61、62、63、64、65或68所示的序列。
  6. 如权利要求1-5中任一所述人源化抗人CD47单克隆抗体或其片段，其特征在于，所述的人源化抗人CD47单抗或其片段具有SEQ ID NO:8、11、12、13、14、15、16、17、18、19、20、21、22或23所示重链可变区，SEQ区ID NO:5所示轻链可变区。
  7. 如权利要求1-6中任一项所述人源化抗人CD47单克隆抗体或其片段，其特征在于，所述单克隆抗体为衍生化抗体，所述衍生化抗体包括在初始单克隆抗体的基础上进行CDRs移植、亲和力成熟、点突变改造和化学修饰获得的抗体，其中，所述化学修饰包括糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、蛋白酶切割、与细胞配体或效应分子连接、活性 反应基团保护和/或封闭。
  8. 如权利要求1-7中任一项所述人源化抗人CD47单克隆抗体或其片段，其特征在于：所述人源化抗人CD47单抗或其片段部分包括Fab、Fab’、F(ab’)2、Fv、scFv或dAb。
  9. 一种多特异性抗体，其特征在于：所述多特异性抗体至少包括一个针对人CD47的特异性的结合位点，所述针对人CD47的特异性结合位点是由权利要求1-8中任一项所述人源化抗人CD47单抗或其片段提供的。
  10. 如权利要求9所述多特异性抗体，其特征在于：所述多特异性抗体包括针对人CD47不同表位的多个特异性结合位点。
  11. 如权利要求9-10中任一项所述多特异性抗体，其特征在于：所述多特异性抗体还包括针对非人CD47靶向的特异性结合位点。
  12. 如权利要求9-11中任一所述多特异性抗体，其特征在于：所述多特异性抗体为双特异性抗体、三特异性抗体或四特异性抗体。
  13. 一种靶向PD-L1和CD47的双特异性抗体或其抗原结合片段，其特征在于，包括两条重链和两条轻链，其中，第一重链可变区源自抗CD47单克隆抗体的重链可变区、其能够与轻链可变区形成CD47结合位点，第二重链可变区源自抗PD-L1单克隆抗体的重链可变区、其能够与轻链可变区形成PD-L1结合位点；
    其中，所述两条轻链的轻链可变区的序列相同，并且，所述轻链可变区是基于抗CD47单克隆抗体的轻链和抗PD-L1单克隆抗体的轻链得到的，且所述轻链可变区与所述第一重链可变区和所述第二重链可变区相匹配，形成的第一可变区结合位点与CD47特意性结合，形成的第二可变区结合位点与CD47PD-L1特意性结合，
    其中，所述抗CD47单克隆抗体为权利要求1-8任一所述的人源化抗人CD47单抗或其片段。
  14. 如权利要求13所述靶向PD-L1和CD47的双特异性抗体或其抗原结合片段，其特征在于，所述抗PD-L1单克隆抗体包括SEQ ID NO:3所示重链可变区和SEQ ID NO:1所示轻链可变区。
  15. 如权利要求13所述靶向PD-L1和CD47的双特异性抗体或其抗原结合片段，其特征在于，所述第二重链可变区具有SEQ ID NO:46所示的CDR1和SEQ ID NO:61、62、63、70、71或72所示的CDR3。
  16. 如权利要求13所述靶向PD-L1和CD47的双特异性抗体或其抗原结合片段，其特征在于，所述轻链可变区具有SEQ ID NO:1、24、25、26、27、28、29、30、31、32,、33或34所示的序列。
  17. 如权利要求13-16任一所述的双特异性抗体或其抗原结合片段，其特征在于，所述 抗体或其抗原结合片段包括但不限于完全抗体、F(ab')2，所述完全抗体为IgG1、IgG2a、IgG2b、IgG3或IgG4型抗体。
  18. 如权利要求13所述的双特异性抗体或其抗原结合片段，其特征在于，所述完全抗体为IgG1或IgG4型。
  19. 如权利要求13-18任一所述的双特异性抗体或其抗原结合片段，其特征在于，所述两条重链的Fc段包括“knobs-into-holes”突变。
  20. 如权利要求13述的双特异性抗体或其抗原结合片段，其特征在于，所述第一重链的Fc段包含H315R突变、所述第二重链的Fc段包括K319E突变，所述Fc段氨基酸突变位点使用Kabat的Eu编号系统。
  21. 一种多核苷酸，其特征在于，编码权利要求1-8任一所述的人源化抗人CD47单抗或其片段，或权利要求9-12任一项所述的多特异性抗体，或权利要求13-20任一项所述的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段。
  22. 载体，其特征在于，包括权利要求21所述的多核苷酸。
  23. 宿主细胞，其特征在于，包括权利要求21所述的多核苷酸或权利要求22所述的载体。
  24. 一种制备抗体或其抗原结合片段的方法，其包括以下步骤：
    (1)在适合表达双特异性抗体或其抗原结合片段的条件下培养权利要求23所述宿主细胞；
    (2)从细胞培养物中分离纯化双特异性抗体或其片段。
  25. 一种组合物，其包含权利要求1-8任一所述的人源化抗人CD47单抗或其片段，或权利要求9-12任一项所述的多特异性抗体，或权利要求13-20任一项所述的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段、权利要求21所述多核苷酸、权利要求22所述载体和权利要求23所述宿主细胞中的至少一种。
  26. 权利要求1-8任一所述的人源化抗人CD47单抗或其片段，或权利要求9-12任一项所述的多特异性抗体，或权利要求13-20任一项所述的靶向PD-L1和CD47的双特异性抗体或其抗原结合片段、权利要求21所述多核苷酸和权利要求25所述组合物在制备治疗肿瘤和/或提高机体免疫应答药物中的用途。
  27. 如权利要求26所述的用途，其特征在于，所述肿瘤包括黑色素瘤、肺癌、肾癌、霍奇金淋巴瘤、头颈鳞癌、尿路上皮癌、急慢性髓系白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤、膀胱癌、卵巢癌、乳腺癌、直肠癌、前列腺癌、肾癌和多发性骨髓瘤。
  28. 治疗患有肿瘤的对象的方法，所述方法包括向所述对象施用治疗有效量的组合物，所述组合物为权利要求要求25所述的组合物。
  29. 如权利要求28所述的方法，其特征在于，所述肿瘤包括黑色素瘤、肺癌、肾癌、霍奇金淋巴瘤、头颈鳞癌、尿路上皮癌、急慢性髓系白血病、急性淋巴细胞白血病、非霍奇金淋巴瘤、膀胱癌、卵巢癌、乳腺癌、直肠癌、前列腺癌、肾癌和多发性骨髓瘤。
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