WO2020098672A1 - 一种融合蛋白及其用途 - Google Patents

一种融合蛋白及其用途 Download PDF

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WO2020098672A1
WO2020098672A1 PCT/CN2019/117856 CN2019117856W WO2020098672A1 WO 2020098672 A1 WO2020098672 A1 WO 2020098672A1 CN 2019117856 W CN2019117856 W CN 2019117856W WO 2020098672 A1 WO2020098672 A1 WO 2020098672A1
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seq
fusion protein
antibody
amino acid
fragment
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PCT/CN2019/117856
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English (en)
French (fr)
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吕明
丁晓然
缪仕伟
谈彬
王学恭
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杭州尚健生物技术有限公司
尚健单抗（北京）生物技术有限公司
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Priority to CN201980075046.0A priority Critical patent/CN113015751A/zh
Priority to BR112021009366-4A priority patent/BR112021009366A2/pt
Priority to EP19884036.5A priority patent/EP3882277A4/en
Priority to KR1020217016172A priority patent/KR20210093265A/ko
Priority to AU2019379207A priority patent/AU2019379207A1/en
Priority to SG11202104912SA priority patent/SG11202104912SA/en
Priority to CA3119400A priority patent/CA3119400A1/en
Priority to JP2021525715A priority patent/JP2022513008A/ja
Priority to US17/293,729 priority patent/US20210317230A1/en
Publication of WO2020098672A1 publication Critical patent/WO2020098672A1/zh

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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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    • C07K2319/00—Fusion polypeptide
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Definitions

  • the present application relates to the field of biomedicine, in particular to a multispecific fusion protein, and also to its use in treating tumors and / or autoimmune diseases.
  • CD47 protein is a transmembrane glycoprotein, which is a member of the immunoglobulin superfamily. In addition to normal tissue cells expressing CD47, many tumor cells overexpress CD47. The combination of CD47 on the surface of tumor cells and SIRP ⁇ on the surface of macrophages prevents macrophages from engulfing tumor cells, which is regarded as a mechanism by which tumors evade immune surveillance. Blocking the interaction between CD47 protein and SIRP ⁇ can inhibit tumor growth.
  • the present application provides a fusion protein comprising a first binding domain that specifically binds to a tumor-associated antigen and a second binding domain that specifically binds to CD47 protein.
  • the present application also provides immunoconjugates comprising the fusion protein; nucleic acid molecules encoding the fusion protein; vectors, compositions and cells capable of containing and / or expressing the fusion protein; and methods of preparing the fusion protein.
  • the fusion protein, immunoconjugate, nucleic acid molecule, carrier, composition and cell of the present application have one or more of the following properties: 1) can specifically bind CD47 protein and tumor-associated antigen at the same time; 2) can specifically block The interaction of CD47 protein with SIRP ⁇ ; 3) can effectively inhibit the growth and / or proliferation of tumors or tumor cells.
  • the present application provides a fusion protein comprising: a first binding domain that specifically binds to a tumor-associated antigen; and a second binding domain that specifically binds to CD47 protein; A mutant of variant 1, which comprises substitution, deletion or addition of amino acid residues at one or more positions from position 33 to position 149 compared to the sequence shown in SEQ ID NO: 50.
  • the mutant comprises amino acid substitutions at one or more amino acid residues selected from the group consisting of R22, I29, I61, V63, E77, Q82, K83, E84, V93, D95, D96 , K98, N100, R107, G109 and V132.
  • the mutant comprises amino acid substitutions at amino acid residues selected from the group consisting of: (1) I61, V63, E77, E84, V93, L96, K98, N100, and V132; (2) I61 , E77, Q82, K83, and E84; (3) I61, V63, K83, E84, and V132; (4) I61, E77, E84, R107, and V132; (5) I61, V63, E77, K83, E84, and N100; (6) I61, E77, Q82, K83, E84, and R107; (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109, and V132; (8) I61, E77, Q82, K83, E84 And V132; (9) I61; (10) I61, D95, L96, G109 and V132; (11) I61, D95, L96, K98, G109 and V132; (12) I61, E77, E84,
  • the mutant comprises one or more amino acid substitutions selected from the group consisting of R22C, I29L, I61L / V / F, V63I, E77I / N / Q / K / H / M / R / N / V / L, Q82S / R / G / N, K83R, E84K / H / D / R / G, V93L / A, D95H / R / E, D96S / T, K98R, N100G / K / D / E, R107N / S, G109R / H and V132L / R / I / S.
  • the mutant comprises an amino acid substitution selected from the group consisting of: (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G, and V132L; (2) I61V, E77N, Q82S, K83R and E84H; (3) I61F, V63I, K83R, E84K and V132I; (4) I61L, E77Q, E84D, R107N and V132I; (5) I61L, V63I, E77K, K83R, E84D and N100G; (6) I61V, E77H, Q82R, K83R, E84H and R107S; (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R and V132R; (8) I61L, E77M, Q82G, K83R, E84D and V132L; (9 ) I61L; (10) I61F,
  • the mutant comprises the amino acid sequence shown in any one of SEQ ID NO: 51-70.
  • the first binding domain comprises an antibody or antigen-binding fragment or variant thereof.
  • the antibody is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2, F (ab) 2, dAb, isolated complementarity determining regions CDR, Fv and scFv.
  • the variant of the antibody or antigen-binding fragment thereof is selected from the group consisting of:
  • the tumor-associated antigens include tumor-associated antigens associated with non-solid tumors and / or solid tumors.
  • the tumor-associated antigen is selected from the group consisting of CD38, AXL, and Trop2.
  • the first binding domain comprises a CD38 antibody or antigen-binding fragment or variant thereof.
  • the CD38 is human CD38.
  • the antibody comprises an antibody heavy chain or fragment thereof, and the antibody heavy chain or fragment thereof comprises HCDR1-3, and the amino acid sequence of the HCDR1-3 is SEQ ID NO: 4, SEQ ID NO : 5 and SEQ ID NO: 6.
  • the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8.
  • the antibody heavy chain or fragment thereof comprises a heavy chain constant region, and the heavy chain constant region comprises IgG.
  • the IgG is selected from the group consisting of IgG1 and IgG4.
  • the antibody heavy chain comprises any one selected from the group consisting of the amino acid sequences shown in the following group: SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: twenty one.
  • the antibody comprises an antibody light chain or fragment thereof, and the antibody light chain or fragment thereof comprises LCDR1-3, and the amino acid sequence of the LCDR1-3 is SEQ ID NO: 1, SEQ ID NO : 2 and SEQ ID NO: 3.
  • the antibody light chain or fragment thereof comprises a light chain variable region VL, and the light chain variable region VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7.
  • the antibody light chain or fragment thereof comprises a light chain constant region, and the light chain constant region thereof comprises Ig ⁇ .
  • the antibody light chain comprises any one selected from the group consisting of the amino acid sequences shown in the following group: SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.
  • the first binding domain comprises an AXL antibody or antigen-binding fragment or variant thereof.
  • the AXL is human AXL.
  • the antibody comprises an antibody heavy chain or fragment thereof, and the antibody heavy chain or fragment thereof comprises HCDR1-3, and the amino acid sequence of the HCDR1-3 is SEQ ID NO: 25, SEQ ID NO : 26 and SEQ ID NO: 27.
  • the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29.
  • the antibody heavy chain or fragment thereof comprises a heavy chain constant region, and the heavy chain constant region comprises IgG.
  • the IgG is selected from the group consisting of IgG1 and IgG4.
  • the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 34.
  • the antibody comprises an antibody light chain or fragment thereof, and the antibody light chain or fragment thereof comprises LCDR1-3, and the amino acid sequence of the LCDR1-3 is SEQ ID NO: 22, SEQ ID NO : 23 and SEQ ID NO: 24.
  • the antibody light chain or fragment thereof comprises a light chain variable region VL, and the light chain variable region VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 28.
  • the antibody light chain or fragment thereof comprises a light chain constant region, and the light chain constant region thereof comprises Ig ⁇ .
  • the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32.
  • the first binding domain comprises Trop2 antibody or antigen-binding fragment or variant thereof.
  • the Trop2 is human Trop2.
  • the antibody comprises an antibody heavy chain or fragment thereof, and the antibody heavy chain or fragment thereof comprises HCDR1-3, and the amino acid sequence of the HCDR1-3 is SEQ ID NO: 39, SEQ ID NO : 40 and SEQ ID NO: 41.
  • the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH, and the heavy chain variable region VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 43.
  • the antibody heavy chain or fragment thereof comprises a heavy chain constant region, and the heavy chain constant region comprises IgG.
  • the IgG is selected from the group consisting of IgG1 and IgG4.
  • the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 48.
  • the antibody comprises an antibody light chain or fragment thereof, and the antibody light chain or fragment thereof comprises LCDR1-3, and the amino acid sequence of the LCDR1-3 is SEQ ID NO: 36, SEQ ID NO : 37 and SEQ ID NO: 38.
  • the antibody light chain or fragment thereof comprises a light chain variable region VL, and the light chain variable region VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 42.
  • the antibody light chain or fragment thereof comprises a light chain constant region, and the light chain constant region thereof comprises Ig ⁇ .
  • the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46.
  • the first binding domain is located at the N-terminus of the second binding domain.
  • the fusion protein further comprises a linker located at the C-terminus of the first binding domain and at the N-terminus of the second binding domain.
  • the linker comprises the amino acid sequence shown in any one of SEQ ID NO: 73-74.
  • the fusion protein contains at least two of the second binding domains. In some embodiments, each of the second binding domains is located at the C-terminus of the first binding domain.
  • the present application provides an immunoconjugate comprising the fusion protein.
  • the present application provides one or more isolated nucleic acid molecules that encode the fusion protein or the immunoconjugate.
  • the present application provides one or more vectors, which contain the nucleic acid molecule.
  • the present application provides a composition comprising the fusion protein, the immunoconjugate, or the nucleic acid molecule, and optionally a pharmaceutically acceptable excipient.
  • the present application provides a cell comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, or the carrier.
  • the present application provides a method for preparing the fusion protein, which includes culturing the cell under conditions enabling the fusion protein to be expressed.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the composition, or the use of the cell in the preparation of medicines , Where the drug is used to treat tumors or autoimmune diseases.
  • the tumor includes non-solid tumors and solid tumors.
  • the tumor includes multiple myeloma, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma, germ cell tumor, sarcoma, epidermal tumor, placental tumor, brain Cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, prostate cancer, intestinal cancer, breast cancer, cervical cancer, ovarian cancer and testicular cancer.
  • the autoimmune disease includes chronic lymphatic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis Syndrome (goodpasture), pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis , Scleroderma and polyarteritis nodosa.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the composition, or the cell, which treats tumors or Immune diseases.
  • the present application provides a method for blocking the interaction between CD47 protein and SIRP ⁇ , which comprises administering an effective amount of the fusion protein, the immunoconjugate to a subject in need, the Nucleic acid molecule, the carrier, the composition, or the cell.
  • the present application provides a method for inhibiting the growth and / or proliferation of tumors or tumor cells, which comprises administering an effective amount of the fusion protein, the immunoconjugate to a subject in need, The nucleic acid molecule, the carrier, the composition, or the cell.
  • Figure 1 shows an example of the structure of the fusion protein described in this application.
  • Figures 2 and 3 show the biological activity of the fusion protein described in this application.
  • 4A and 4B show the biological activity of the fusion protein described in this application.
  • FIG. 5 shows a schematic diagram of the method for detecting the interaction between the fusion protein described in this application and CD38 and CD47.
  • Figure 6 shows the biological activity of the fusion protein described in this application.
  • FIG. 7 is a schematic diagram showing the principle of the method for detecting the interaction between the fusion protein described in this application and CD38 and CD47.
  • FIGS 8 and 9 show the biological activity of the fusion protein described in this application.
  • Figure 10 shows that the fusion protein described in this application blocks the interaction between CD47 protein and SIRP ⁇ .
  • 11A-11B, 12A-12B and 13A-13B show the binding activity of the fusion protein of the present application and the corresponding antigen.
  • Figure 14 shows tumor imaging results of mice in different treatment groups.
  • Figure 15 shows the average fluorescence intensity of tumors in mice of different treatment groups.
  • Figure 16 shows the survival rate of mice in different treatment groups.
  • fusion protein generally refers to a protein resulting from the fusion of two or more proteins or polypeptides.
  • the fusion protein can be artificially prepared by recombinant DNA technology.
  • genes or nucleic acid molecules encoding the two or more proteins or polypeptides may be linked to each other to form a fusion gene or fusion nucleic acid molecule, and the fusion gene or fusion nucleic acid molecule may encode the fusion protein.
  • the translation of the fusion gene may produce a single polypeptide, which may have the property of at least one or even each of the two or more proteins or polypeptides before fusion.
  • the term "specific binding” generally refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen that produces the antibody.
  • a particular antigen-specific antibody means an affinity (KD) ⁇ 10 -5 M (such as 10 -6 M, 10 - 7 M , 10 -8 M, 10 -9 M, 10 -10 M , etc.) binding of the antigen, Where KD refers to the ratio of dissociation rate to binding rate (koff / kon), which can be determined by methods familiar to those skilled in the art.
  • binding domain is generally a domain that can specifically bind and / or recognize a specific epitope on a target (eg, antigen).
  • domain generally refers to a region of closely spherical structures in protein subunit structures that are clearly separated.
  • a polypeptide chain can first form a regular secondary structure by adjacent amino acid residues in certain regions, and then, it can be assembled from adjacent secondary structure fragments to form a super secondary structure.
  • the polypeptide chain can be folded into an approximately spherical tertiary structure.
  • the polypeptide chain can often be formed by the association of two or more spatially distinct, relatively independent regional structures to form a tertiary structure. This relatively independent regional structure Can be called a domain.
  • first and second in the terms “first binding domain” and “second binding domain” may be used only for distinguishing in description.
  • CD47 protein generally refers to an integrin-related protein (IAP), which is a multiple transmembrane receptor belonging to the immunoglobulin superfamily.
  • IAP integrin-related protein
  • CD47 protein can be combined with membrane integrins and its ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRP ⁇ ).
  • TSP-1 membrane integrins and its ligands thrombospondin-1
  • SIRP ⁇ signal-regulatory protein alpha
  • CD47 protein is widely expressed on the surface of cell membrane.
  • the CD47 protein may include any variant, isotype, and species homolog of human CD47.
  • the amino acid sequence of human CD47 protein is listed as CEJ95640.1 in GenBank.
  • the CD47 protein can be naturally expressed by cells or expressed on cells transfected with the CD47 gene.
  • SIRP ⁇ generally refers to a regulatory membrane glycoprotein from the SIRP family, which can serve as a ligand for the CD47 protein.
  • the SIRP ⁇ may include human SIRP ⁇ .
  • the SIRP ⁇ variant 2 differs from the SIRP ⁇ variant 1 by 13 amino acids, and its amino acid sequence is listed as CAA71403.1 in GenBank.
  • SIRP ⁇ variant 1 generally refers to the SIRP ⁇ protein whose amino acid sequence is listed as NCBI RefSeq NP_542970.1 (residues 31-504 constitute the mature form), in which case the amino acid sequence of SIRP ⁇ variant 1 is as SEQ ID NO: 50.
  • variant generally refers to a proteinaceous molecule having sequence homology to a protein that does not possess any mutations / modifications, which retains at least a portion of the therapeutic and / or biological activity of the biologically active protein.
  • the variant protein may share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% compared to the reference bioactive protein Amino acid sequence identity.
  • variants may include proteins that have been intentionally modified (eg, by site-directed mutagenesis, synthesis of a coding gene, insertion, or accidentally by mutation).
  • antibody generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • immunoglobulin genes can include ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ constant region genes, as well as countless immunoglobulin variable region genes.
  • light chains can be classified as ⁇ or ⁇ , which can define immunoglobulin types: Ig ⁇ and Ig ⁇ , respectively.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , which in turn define immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively.
  • an antibody may have a structural unit containing tetramers, each tetramer may be composed of two pairs of the same polypeptide chain, each pair has a "light” chain (about 25kD) and a "heavy chain” (about 50-70kD ),
  • the N-terminus of each member may define a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition.
  • the terms "light chain variable region (VL)” and “heavy chain variable region (VH)” generally refer to the light chain and heavy chain variable region regions, respectively.
  • Antibodies can exist as intact immunoglobulins or as many well-characterized fragments produced by digestion with various peptidases or de novo expression.
  • the term "antigen-binding fragment” generally refers to one or more parts of a full-length antibody that substantially retain the ability to bind the same antigen (eg, CD38) to which the antibody binds, capable of binding the full-length antibody Competition for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), and the full text of which is incorporated by reference into this application. It may be by recombinant DNA technology or by whole antibodies Enzymatic or chemical fragmentation of the antigen to produce antigen-binding fragments.
  • antigen-binding fragments include Fab, Fab ', F (ab') 2, (Fab) 2, Fd, Fv, dAb and complementarity determining region (CDR) fragments , Single-chain antibodies (eg, scFv), chimeric antibodies, diabodies (diabody), and polypeptides that contain at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Conventions known to those skilled in the art may be used Technology (for example, recombinant DNA technology or enzymatic or chemical fragmentation) to obtain antigen-binding fragments of antibodies from a given antibody, and to specifically screen for antigen-binding fragments of antibodies in the same manner as for intact antibodies.
  • Pepsin can digest antibodies below the disulfide bond in the hinge region to produce F (ab ') 2.
  • Fab generally refers to antibody fragments composed of VL, VH, CL, and CH1 domains.
  • Fab ' generally refers to an antibody fragment having several additional residues at the carboxy terminus of the CH1 domain compared to the Fab fragment.
  • Fab ' may include one or more cysteines from the hinge region of the antibody.
  • F (ab) 2 generally refers to an antigen-binding fragment obtained from a pair of Fab fragments linked by cysteine.
  • dAb fragment generally refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341: 544-546 (1989)).
  • complementarity determining region CDR generally refers to the three hypervariable regions (HVR) of the light chain variable region (VL) and the heavy chain variable region (VH). It forms precise complementarity with antigenic determinants, so hypervariable regions are also called complementarity determining regions.
  • Fv fragment generally refers to an antibody fragment consisting of the VL and VH domains of one arm of an antibody.
  • scFv generally refers to a molecule composed of an antibody heavy chain variable region and a light chain variable region connected by a short peptide linker, also known as a single chain antibody.
  • the term "monoclonal antibody” generally refers to a group of substantially homologous antibodies, and each antibody included in the group may be the same except for possible naturally occurring mutations in trace amounts. Monoclonal antibodies are highly specific and directly target a single antigenic site. In addition, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant modifier on the antigen "monoclonal” is not interpreted as requiring any special method Produce antibodies.
  • the monoclonal antibody may be prepared by hybridoma technology or produced by using recombinant DNA methods in bacteria, eukaryotic animals or plant cells, or may be obtained from a phage antibody library using, for example, Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).
  • chimeric antibody generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific category, and The rest of the chain is homologous to the corresponding sequence in another species.
  • the variable regions of the light and heavy chains are derived from the variable regions of antibodies of one animal species (such as mouse, rat, etc.), while the constant part is homologous to the antibody sequence from another species (such as human).
  • non-human B cells or hybridoma cells can be used to generate variable regions, while the constant regions combined with them are derived from humans.
  • variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined with it.
  • the constant region of the chimeric antibody can be derived from humans, the chimeric antibody will be less likely to elicit an immune response when injected by the antibody than when the constant region is an antibody of non-human origin.
  • humanized antibody generally refers to the reduction in the immunogenicity of antibodies, immunoglobulin binding proteins and polypeptides derived from non-human species (eg, mouse or rat) to the human body while still retaining An engineered antibody with the antigen-binding properties of the original antibody.
  • genetic engineering techniques can be used to prepare humanized antibodies, and CDR grafting (Jones et al., Nature 321: 522 (1986)) and its variants can be used; including “reshaping” (Verhoeyen, et al.
  • the term "fully human antibody” generally refers to an antibody obtained by expressing a gene encoding a human antibody in a genetically engineered antibody gene-deficient animal.
  • the human antibody gene can be transferred to all genetically engineered antibody gene-deficient animals through transgenic or chromosomal technology to allow the animal to express human antibodies
  • sequence homology generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences.
  • a program eg Emboss Needle or BestFit
  • the default settings can be used, or an appropriate scoring matrix (such as blosum45 or blosum80) can be selected To optimize identity, similarity, or homology scores.
  • homologous polynucleotides are those sequences that hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, and even 100% sequence identity.
  • homologous polypeptides When aligning sequences of considerable length, homologous polypeptides have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity, or have at least 99% sequence Identity.
  • CD38 CD38 protein
  • CD38 antigen any variants, isotypes and species homologs of human CD38, which can be naturally produced by cells Express or on cells transfected with CD38 gene.
  • Art-recognized synonyms for CD38 include ADP ribosyl cyclase 1, cADPr hydrolase 1, Cd38-rs1, cyclic ADP-ribose hydrolase 1, I-19, and NIM-R5 antigens.
  • CD38 can be highly expressed on the surface of myeloma cells.
  • AXL AXL protein
  • AXL antigen a protein belonging to the tyrosine kinase family, which can be composed of a common ligand Gas6.
  • AXL can be used as a marker of cancer cell deterioration.
  • the AXL may be human AXL, and its amino acid sequence is listed as AAH32229 in GenBank.
  • Trop2 generally refers to tumor-associated calcium signal transducer 2 (tumor-associated calcium signal transducer 2), which may be referred to as TACSTD2, EGP-1, GA733-1, GP50, or the like.
  • Trop2 can be located on the cell membrane in the cell, and its positive staining and coloring sites can be mainly on the cell membrane. In normal tissues, Trop2 expression is very low, but it is overexpressed in tumor cells, so it can be used as a target for tumor treatment.
  • the Trop2 may be human Trop2, and its amino acid sequence is listed as NP_002344 in GenBank.
  • the amino group is connected to another carboxyl group in the polypeptide chain to make it a chain, but at the two ends of the protein, the amino acid residues that do not form peptide bonds are left, respectively, carrying free The end of the amino acid polypeptide chain and the end of the carboxyl carrying polypeptide chain.
  • N-terminal generally refers to the end of a polypeptide chain whose amino acid residue carries a free amino group.
  • C-terminal generally refers to the end of a polypeptide chain in which amino acid residues carry a free carboxyl group.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from its natural environment or artificially synthesized, or analogs thereof.
  • the term “immunoconjugate” generally refers to a polypeptide molecule with immunological function conjugated with one or more heterologous molecules (including but not limited to cytotoxin).
  • heterologous molecules including but not limited to cytotoxin
  • conjugation and “linkage” and “fusion” are used interchangeably in this application, and generally refers to connecting two or more chemical elements, sequences or components together, for example by Including chemical conjugation or recombination means.
  • the heterologous molecule may be a cytotoxin, a chemotherapy drug, or the like.
  • the immunoconjugate can be obtained by conjugating the fusion protein described in this application with one or more heterologous molecules (for example, cytotoxin).
  • vector refers to a nucleic acid delivery vehicle in which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material element carried in the host cell is expressed.
  • vectors include: plasmids; phagemids; Coase plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC); phages such as lambda phage or M13 phage And animal viruses.
  • Animal virus types used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), pox viruses, baculoviruses, papilloma viruses, and papilloma Viruses (such as SV40).
  • a vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may contain an origin of replication.
  • the carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.
  • tumors generally refers to a neoplasm formed by the proliferation of local tissues and cells under the action of various tumorigenic factors (for example, cells or their constituent parts) in mammals.
  • tumors may include solid tumors and non-solid tumors.
  • Solid tumors may include gliomas, germ cell tumors, sarcomas, epidermal tumors, placental tumors, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, Prostate cancer, bowel cancer, breast cancer, cervical cancer, ovarian cancer and testicular cancer.
  • non-solid tumors may include multiple myeloma, leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma.
  • lymphoma generally refers to a malignant tumor of the lymphatic system. Lymphoma can occur because lymph node cells or lymphocytes begin to multiply uncontrollably and produce cancer cells with abnormal capabilities that can invade other tissues throughout the body. Patient signs and symptoms can include enlarged lymph nodes, fever, night sweats, weight loss, or itching, and they continue to feel tired. There are many subtypes of lymphoma, and the two main types can be Hodgkin lymphoma and non-Hodgkin lymphoma. In the present application, the term “Hodgkin's lymphoma” (HL) generally refers to a lymphoma produced by lymphocyte-like leukocytes.
  • Non-Hodgkin lymphoma Non-Hodgkin lymphoma, NHL generally refers to other types of lymphoma than Hodgkin lymphoma.
  • leukemia generally refers to a malignant proliferative disease of the hematopoietic system, generally refers to a type of disease caused by the proliferation and accumulation of leukemia cells in large quantities.
  • Clonal leukemia cells proliferate and accumulate in the bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, differentiation differentiation, and blocked apoptosis, and infiltrate other non-hematopoietic tissues and organs while inhibiting normal hematopoietic function.
  • Clinically, varying degrees of anemia, hemorrhage, fever, infection, swelling of the liver, spleen, and lymph nodes and bone pain are seen.
  • multiple myeloma generally refers to a malignant tumor in which the abnormal proliferation of plasma cells causes invasion of the bone marrow. Multiple myeloma causes cancer cells to accumulate in the bone marrow, allowing healthy blood cells to be eliminated. Cancer cells produce abnormal proteins that can cause complications instead of normal antibodies. The incidence of multiple myeloma can increase among the elderly. Its chemotherapy regimen has low cell proliferation rate and strong drug resistance, which limits its chemotherapy effect. More than 90% of patients with multiple myeloma have chemotherapy resistance.
  • Raji cell generally refers to a continuous human cell line capable of producing Epstein-Barr virus strain.
  • the virus will transform umbilical cord lymphocytes and induce early antigens in Raji cells.
  • Raji cells can be widely used as a transfection host, and can also be used to understand hematopoietic cells and other cell malignancies.
  • Raji cells have several receptors that express certain complement components and Fc receptors for immunoglobulin G, so they can also be used to detect immune complexes.
  • autoimmune disease generally refers to a disease caused by the body's immune response to self-antigens resulting in damage to its own tissues.
  • Autoimmune diseases can be divided into two categories, organ-specific autoimmune diseases and systemic autoimmune diseases.
  • Organ-specific autoimmune diseases can refer to the pathological damage and dysfunction of tissues and organs is limited to an organ targeted by antibodies or sensitized lymphocytes, which may include chronic lymphatic thyroiditis, hyperthyroidism, insulin-dependent diabetes, severe disease Muscle weakness, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis syndrome (goodpasture syndrome), pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal cord Sclerosis and acute idiopathic polyneuritis.
  • Systemic autoimmune diseases can be caused by the extensive deposition of antigen-antibody complexes on the blood vessel wall and other causes of systemic multiple organ damage.
  • systemic autoimmune diseases may include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, and polyarteritis nodosa.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR killing activity
  • the term "about” generally refers to a range of 0.5% -10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a fusion protein, which may include a first binding domain and a second binding domain.
  • the first binding domain can specifically bind to a tumor-associated antigen; the second binding domain can specifically bind to a CD47 protein, and the second binding domain can comprise a mutant of human SIRP ⁇ variant 1, the mutant and SEQ ID: NO:
  • One or more of the sequences shown in position 33 to position 149 (for example, 1-2, 1-3, 1-4, 1-5, 1- Six, 1-7, 1-8, 1-9, 1-10 or more) positions contain substitutions, deletions or additions of amino acid residues.
  • the fusion protein described in the present application can specifically bind to tumor-associated antigen and CD47 protein at the same time, thereby playing the role of treating tumor and / or autoimmune diseases.
  • first binding domain generally refers to a domain that can specifically bind to a tumor-associated antigen.
  • second binding domain generally refers to a domain that can specifically bind to CD47 protein.
  • the tumor-associated antigens may include tumor-associated antigens associated with non-solid tumors and / or solid tumors.
  • the tumor-associated antigen may be selected from the group consisting of CD38, AXL, and Trop2.
  • the mutant for example, a mutant of human SIRP ⁇ variant 1 that specifically binds to CD47 protein
  • the mutant is selected from one or more of the following groups (for example, 1-2, 1-3, 1 -4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid residues contain amino acid substitutions: R22, I29, I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109 and V132.
  • the position of the amino acid residue in the amino acid substitution is the residue number determined based on the amino acid sequence shown in SEQ ID NO: 50.
  • amino acid substitution Xn refers to an amino acid substitution at residue X corresponding to the nth position in the amino acid sequence shown in SEQ ID NO: 50, where n is a positive integer and X is any amino acid residue.
  • amino acid substitution I61 means that an amino acid substitution occurs at residue I corresponding to position 61 in the amino acid sequence shown in SEQ ID NO: 50.
  • the amino acid substitution may be a non-conservative substitution.
  • the non-conservative substitution may include changing the amino acid residues in the target protein or polypeptide in a non-conservative form, for example, changing amino acid residues having a certain side chain size or a certain characteristic (eg, hydrophilicity) to have different Amino acid residues with side chain size or different characteristics (eg, hydrophobicity).
  • the amino acid substitution may also be a conservative substitution.
  • the conservative substitution may include conservatively changing the amino acid residues in the target protein or polypeptide, for example, changing amino acid residues having a certain side chain size or a certain characteristic (eg, hydrophilicity) to have the same or similar Amino acid residues of side chain size or the same or similar characteristics (eg, still hydrophilic).
  • Such conservative substitutions usually do not greatly affect the structure or function of the protein produced.
  • the amino acid sequence variant as the fusion protein or fragment thereof may include a mutant that does not significantly change the structure or function of the protein (for example, a mutant that blocks CD47 and human SIRP ⁇ variant 1 that specifically binds to the CD47 protein) Of conservative amino acid substitutions.
  • amino acid groups with non-polar side chains alanine, valine, leucine, Isoleucine, proline, phenylalanine, tryptophan and methionine.
  • Uncharged amino acid groups with polar side chains glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • Negatively charged amino acid groups with polar side chains aspartic acid and glutamic acid.
  • Positively charged basic amino acids lysine, arginine and histidine.
  • Amino acids with phenyl groups phenylalanine, tryptophan and tyrosine.
  • the mutant may include amino acid substitutions at amino acid residues selected from the group consisting of: (1) I61, V63, E77, E84, V93, L96, K98, N100, and V132; (2) I61, E77, Q82, K83 and E84; (3) I61, V63, K83, E84 and V132; (4) I61, E77, E84, R107 and V132; (5) I61, V63, E77, K83, E84 and N100; ( 6) I61, E77, Q82, K83, E84 and R107; (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109 and V132; (8) I61, E77, Q82, K83, E84 and V132; (9) I61; (10) I61, D95, L96, G109 and V132; (11) I61, D95, L96, K98, G109 and V132; (12) I61, E77, E84, V93, R107 and
  • the mutant may comprise one or more selected from the group (for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1- 7, 1-8, 1-9, 1-10 or more) amino acid substitutions: R22C, I29L, I61L / V / F, V63I, E77I / N / Q / K / H / M / R / N / V / L, Q82S / R / G / N, K83R, E84K / H / D / R / G, V93L / A, D95H / R / E, D96S / T, K98R, N100G / K / D / E , R107N / S, G109R / H and V132L / R / I / S.
  • group for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1- 7, 1-8, 1-9, 1-10 or more amino acid substitutions: R22C, I29L, I61L /
  • the amino acid substitution "XnY / Z” means that the residue X corresponding to the nth position in the amino acid sequence shown in SEQ ID NO: 50 is substituted with amino acid residue Y or amino acid residue Z, where n is positive
  • the integers, X, Y, and Z are each independently an abbreviation for any amino acid residue, and X is different from Y or Z.
  • the amino acid substitution "I61L / V / F" means that residue I corresponding to position 61 in the amino acid sequence shown in SEQ ID NO: 50 is substituted with amino acid residues L, V, or F.
  • the mutant may comprise an amino acid substitution selected from the group consisting of: (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G, and V132L; (2) I61V, E77N, Q82S, K83R And E84H; (3) I61F, V63I, K83R, E84K and V132I; (4) I61L, E77Q, E84D, R107N and V132I; (5) I61L, V63I, E77K, K83R, E84D and N100G; (6) I61V, E77H , Q82R, K83R, E84H and R107S; (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R and V132R; (8) I61L, E77M, Q82G, K83R, E84D and V132L; (9) I61L; (10) I61F,
  • the above (1) -Mutants of SIRP ⁇ variant 1 of the amino acid substitution group of (20) can be named M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111 in sequence , M122, M126, M130, M135 and M145.
  • the mutant of SIRP ⁇ variant 1 may sequentially include the amino acid sequence shown in SEQ ID NO: 51-SEQ ID NO: 70.
  • the mutant of SIRP ⁇ variant 1 is M91, and the mutant of SIRP ⁇ variant 1 comprises the amino acid sequence shown in SEQ ID NO: 62.
  • the first binding domain that specifically binds to tumor-associated antigens
  • the first binding domain may comprise an antibody or antigen-binding fragment or variant thereof.
  • the antibody may be selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab ', (Fab') 2, F (ab) 2, dAb, isolated complementarity determining regions CDR, Fv and scFv.
  • the variant of the antibody or antigen-binding fragment thereof may be substituted, deleted, or added one or more (eg, 1-2, 1 -3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid protein or polypeptide.
  • the variant of the antibody or antigen-binding fragment thereof may have at least 90% (eg, for example, at least 90%, at least 91%, at least 92%, at least 93%) with the antibody or the antigen-binding fragment thereof , At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence homology protein or polypeptide.
  • the antibodies or antigen-binding fragments or variants described in this application can specifically bind to CD38 protein to kill tumor cells and / or inhibit tumor growth.
  • the tumor may include a CD38 positive tumor.
  • the CD38 positive tumor may be selected from the group consisting of myeloma, lymphoma and leukemia.
  • the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma.
  • the cells of the tumor may be selected from the group consisting of Raji cells, Daudi cells, Ramos cells, and RPMI8226 cells.
  • the antibody, antigen-binding fragment or variant thereof can kill multiple myeloma, lymphoma, leukemia, non-Hodgkin lymphoma and Hodgkin lymphoma cells or inhibit multiple myeloma, lymphoma , Leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma growth.
  • the CD38 protein described in this application may be a human CD38 protein or a functional fragment thereof.
  • the CD38 protein may not be the mouse CD38 protein, or may not be the rat CD38 protein.
  • the antibodies, antigen-binding fragments or variants thereof described herein do not substantially bind to mouse CD38 protein or rat CD38 protein.
  • the antibody, antigen-binding fragment or variant thereof described herein can compete with the reference antibody for binding to the CD38 protein.
  • the reference antibody may comprise a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the reference antibody may include LCDR1-3, and the amino acid sequence of the LCDR1-3 is SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in this order.
  • the heavy chain variable region of the reference antibody may include HCDR1-3, and the amino acid sequences of the HCDR1-3 are SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in this order.
  • the amino acid sequence of the light chain variable region of the reference antibody may be: SEQ ID NO: 7
  • the amino acid sequence of the heavy chain variable region of the reference antibody may be: SEQ ID NO: 8.
  • the light chain of the reference antibody may include any one selected from the group consisting of the amino acid sequences shown in the following group: SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20
  • the heavy chain of the reference antibody may comprise any one selected from the amino acid sequence shown in the following group: SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: twenty one.
  • the light chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 11, and the heavy chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 13.
  • the light chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 16 and the heavy chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 17.
  • the light chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 18, and the heavy chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 19.
  • the light chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 20, and the heavy chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 21.
  • the antibodies, antigen-binding fragments or variants thereof described herein may comprise antibody light chains or fragments thereof.
  • the antibody light chain or a fragment thereof may include an Ig ⁇ constant region, for example, may contain a human Ig ⁇ constant region.
  • the antibody light chain or a fragment thereof may include LCDR1, and the LCDR1 may include the following amino acid sequence: SEQ ID NO: 1.
  • the antibody light chain or a fragment thereof may include LCDR2, and the LCDR2 may include the following amino acid sequence: SEQ ID NO: 2.
  • the antibody light chain or a fragment thereof may include LCDR3, and the LCDR3 may include the following amino acid sequence: SEQ ID NO: 3.
  • the light chain or fragment of the antibody of the present application may include a light chain variable region VL, and the amino acid sequence of the light chain variable region VL may be: SEQ ID NO: 7.
  • the antibody light chain or fragment thereof may comprise any one selected from the group consisting of the amino acid sequences shown in the following group: SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.
  • the antibody or antigen-binding fragment thereof described herein may comprise an antibody heavy chain or a fragment thereof.
  • the antibody heavy chain or fragment thereof also contains a human constant region.
  • the human constant region may include a human IgG constant region.
  • the IgG constant region may comprise human IgG1 constant region or IgG4.
  • the antibody heavy chain or fragment thereof may include HCDR1, and the HCDR1 may include the following amino acid sequence: SEQ ID NO: 4.
  • the antibody heavy chain or fragment thereof may include HCDR2, and the HCDR2 may include the following amino acid sequence: SEQ ID NO: 5.
  • the antibody heavy chain or a fragment thereof may include HCDR3, and the HCDR3 may include the following amino acid sequence: SEQ ID NO: 6.
  • the antibody heavy chain or a fragment thereof may include a heavy chain variable region VH, and the heavy chain variable region VH may include the following amino acid sequence: SEQ ID NO: 8.
  • the antibody heavy chain may comprise any one selected from the group consisting of the amino acid sequences shown in the following group: SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO :twenty one.
  • the amino acid sequence of LCDR1 in the antibody or antigen-binding fragment thereof described herein may include SEQ ID NO: 1; the amino acid sequence of LCDR2 may include SEQ ID NO: 2; the amino acid sequence of LCDR3 may include SEQ ID NO: 3; and the amino acid sequence of HCDR1 may include SEQ ID NO: 4 or; the amino acid sequence of HCDR2 may include SEQ ID NO: 5; the amino acid sequence of HCDR3 may include SEQ ID NO: 6.
  • the antibody or antigen-binding fragment thereof may include the antibody SG003 or the antibody having the same LCDR1-3 and HCDR1-3 as it.
  • the light chain of the antibody or antigen-binding fragment thereof described herein may include a light chain variable region, and the amino acid sequence of the light chain variable region may include SEQ ID NO: 7; and wherein the heavy The chain may include a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 8.
  • the antibody or antigen-binding fragment thereof may include the antibody SG003 or the antibody having the same light chain variable region and heavy chain variable region.
  • the antibody or antigen-binding fragment thereof described herein may include a light chain and a heavy chain, and the light chain amino acid sequence of the light chain is shown in SEQ ID NO: 11 and the heavy chain amino acid sequence As shown in SEQ ID NO: 13.
  • the antibody or antigen-binding fragment thereof may include the antibody SG003 or the same light chain and heavy chain amino acid sequences as the antibody SG003.
  • the antibody described in this application may be SG003.
  • the amino acid sequence of LCDR1-3 of antibody SG003 is shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 7; the amino acid sequence of HCDR1-3 As shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 8; the amino acid sequence of the light chain is shown in SEQ ID NO: 11; heavy The amino acid sequence of the chain is shown in SEQ ID NO: 13.
  • the antibodies or antigen-binding fragments or variants described herein may also contain one or more random mutations (for example, one or more, such as one or more) in the amino acid sequence of the light chain and / or heavy chain of SG003 Amino acid substitutions).
  • the antibody, antigen-binding fragment or variant thereof may contain one or more random mutations (e.g., one or more) at one or more positions in the framework region L-FR1-4 of the light chain variable region of SG003 , Such as one or several amino acid substitutions), and / or one or more random mutations (eg, one or more) at one or more positions in the framework region H-FR1-4 of the heavy chain variable region of SG003 , Such as one or several amino acid substitutions).
  • the light chain of the antibody or antigen-binding fragment or variant thereof may contain any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20; and / or the heavy chain of the antibody or antigen-binding fragment or variant thereof may comprise any one selected from the amino acid sequence shown in the following group: SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 21.
  • the CD38 antibody or its antigen-binding fragment or variant after random mutation still has the ability to specifically bind to human CD38 protein.
  • the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof described herein includes SEQ ID NO: 11; and the amino acid sequence of its heavy chain includes SEQ ID NO: 13; or as described in this application
  • the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof includes SEQ ID NO: 16; and the amino acid sequence of the heavy chain includes SEQ ID NO: 17; or, the light chain of the antibody or antigen-binding fragment described herein
  • the amino acid sequence of SEQ ID includes NO: 18; and the amino acid sequence of its heavy chain includes SEQ ID NO: 19; or, the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof described in this application includes SEQ ID NO: 20; And the amino acid sequence of its heavy chain includes SEQ ID NO: 21.
  • the antibodies or antigen-binding fragments or variants described herein can kill tumor cells and / or inhibit tumor growth by specifically binding to AXL.
  • the AXL may be human AXL.
  • the antibody, antigen-binding fragment or variant thereof described in this application can compete with the reference antibody for binding to the AXL protein.
  • the reference antibody may comprise a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the reference antibody may include LCDR1-3, and the amino acid sequence of the LCDR1-3 is SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 in this order.
  • the heavy chain variable region of the reference antibody may include HCDR1-3, and the amino acid sequences of the HCDR1-3 are SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 in this order.
  • amino acid sequence of the light chain variable region of the reference antibody may be: SEQ ID NO: 28, and the amino acid sequence of the heavy chain variable region of the reference antibody may be: SEQ ID NO: 29.
  • amino acid sequence of the light chain of the reference antibody may be: SEQ ID NO: 32; and the amino acid sequence of the heavy chain of the reference antibody may be: SEQ ID NO: 34.
  • the antibodies, antigen-binding fragments or variants thereof described herein may comprise antibody light chains or fragments thereof.
  • the antibody light chain or a fragment thereof may include an Ig ⁇ constant region, for example, may contain a human Ig ⁇ constant region.
  • the antibody light chain or a fragment thereof may include LCDR1, and the LCDR1 may include the following amino acid sequence: SEQ ID NO: 22.
  • the antibody light chain or a fragment thereof may include LCDR2, and the LCDR2 may include the following amino acid sequence: SEQ ID NO: 23.
  • the antibody light chain or a fragment thereof may include LCDR3, and the LCDR3 may include the following amino acid sequence: SEQ ID NO: 24.
  • the light chain or fragment of the antibody of the present application may include a light chain variable region VL, and the amino acid sequence of the light chain variable region VL may be: SEQ ID NO: 28. In some embodiments, the amino acid sequence of the antibody light chain or fragment thereof may be: SEQ ID NO: 32.
  • the antibody or antigen-binding fragment thereof described herein may comprise an antibody heavy chain or a fragment thereof.
  • the antibody heavy chain or fragment thereof also contains a human constant region.
  • the human constant region may include a human IgG constant region.
  • the IgG constant region may comprise human IgG1 constant region or IgG4.
  • the antibody heavy chain or fragment thereof may include HCDR1, and the HCDR1 may include the following amino acid sequence: SEQ ID NO: 25.
  • the antibody heavy chain or a fragment thereof may include HCDR2, and the HCDR2 may include the following amino acid sequence: SEQ ID NO: 26.
  • the antibody heavy chain or fragment thereof may include HCDR3, and the HCDR3 may include the following amino acid sequence: SEQ ID NO: 27.
  • the antibody heavy chain or a fragment thereof may include a heavy chain variable region VH, and the amino acid sequence of the heavy chain variable region VH may be: SEQ ID NO: 29. In some embodiments, the amino acid sequence of the antibody heavy chain or fragment thereof may be: SEQ ID NO: 34.
  • the amino acid sequence of LCDR1 in the antibody or antigen-binding fragment thereof described herein may include SEQ ID NO: 22 or its variant; the amino acid sequence of LCDR2 may include SEQ ID NO: 23 or its variant ; The amino acid sequence of LCDR3 may include SEQ ID NO: 24 or its variant; and the amino acid sequence of HCDR1 may include SEQ ID NO: 25 or its variant; the amino acid sequence of HCDR2 may include SEQ ID NO: 26 or its variant; The amino acid sequence of HCDR3 may include SEQ ID NO: 27 or a variant thereof.
  • the antibody or antigen-binding fragment thereof may include antibody C6G12 or an antibody having the same LCDR1-3 and HCDR1-3 as it.
  • the light chain of the antibody or antigen-binding fragment thereof described herein may include a light chain variable region, and the amino acid sequence of the light chain variable region may include SEQ ID NO: 28 or a variant thereof ; And wherein the heavy chain may comprise a heavy chain variable region, the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 29 or a variant thereof.
  • the antibody or antigen-binding fragment thereof may include antibody C6G12 or an antibody having the same light chain variable region and heavy chain variable region.
  • the antibody or antigen-binding fragment thereof described herein may comprise a light chain and a heavy chain, the light chain amino acid sequence of the light chain is shown in SEQ ID NO: 32 and the heavy chain amino acid sequence As shown in SEQ ID NO: 34.
  • the antibody or antigen-binding fragment thereof may include the antibody C6G12 or have the same light chain and heavy chain amino acid sequences as the antibody.
  • the antibody described in this application may be C6G12.
  • the amino acid sequence of LCDR1-3 of antibody C6G12 is shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 28; the amino acid sequence of HCDR1-3 As shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 29; the amino acid sequence of the light chain is shown in SEQ ID NO: 32; heavy The amino acid sequence of the chain is shown in SEQ ID NO: 34.
  • the antibodies or antigen-binding fragments or variants described in this application can specifically bind to Trop2 to kill tumor cells and / or inhibit tumor growth.
  • the Trop2 may be human Trop2.
  • the antibody, antigen-binding fragment or variant thereof described in this application can compete with the reference antibody for binding to the Trop2 protein.
  • the reference antibody may comprise a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the reference antibody may include LCDR1-3, and the amino acid sequence of the LCDR1-3 is SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38 in this order.
  • the heavy chain variable region of the reference antibody may include HCDR1-3, and the amino acid sequences of the HCDR1-3 are SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41 in this order.
  • the amino acid sequence of the light chain variable region of the reference antibody may be: SEQ ID NO: 42, and the amino acid sequence of the heavy chain variable region of the reference antibody may be: SEQ ID NO: 43.
  • the amino acid sequence of the light chain of the reference antibody may be: SEQ ID NO: 46; and the amino acid sequence of the heavy chain of the reference antibody may be SEQ ID NO: 48.
  • the light chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 46
  • the heavy chain of the reference antibody may include the amino acid sequence shown in SEQ ID NO: 48.
  • the antibodies, antigen-binding fragments or variants thereof described herein may comprise antibody light chains or fragments thereof.
  • the antibody light chain or a fragment thereof may include an Ig ⁇ constant region, for example, may contain a human Ig ⁇ constant region.
  • the antibody light chain or a fragment thereof may include LCDR1, and the LCDR1 may include the following amino acid sequence: SEQ ID NO: 36.
  • the antibody light chain or a fragment thereof may include LCDR2, and the LCDR2 may include the following amino acid sequence: SEQ ID NO: 37.
  • the antibody light chain or a fragment thereof may include LCDR3, and the LCDR3 may include the following amino acid sequence: SEQ ID NO: 38.
  • the light chain or fragment thereof of the antibody of the present application may include a light chain variable region VL, and the light chain variable region VL may include the following amino acid sequence: SEQ ID NO: 42.
  • the amino acid sequence of the antibody light chain or fragment thereof may be: SEQ ID NO: 46.
  • the antibody or antigen-binding fragment thereof described herein may comprise an antibody heavy chain or a fragment thereof.
  • the antibody heavy chain or fragment thereof also contains a human constant region.
  • the human constant region may include a human IgG constant region.
  • the IgG constant region may comprise human IgG1 constant region or IgG4.
  • the antibody heavy chain or fragment thereof may include HCDR1, and the HCDR1 may include the following amino acid sequence: SEQ ID NO: 39.
  • the antibody heavy chain or fragment thereof may include HCDR2, and the HCDR2 may include the following amino acid sequence: SEQ ID NO: 40.
  • the antibody heavy chain or fragment thereof may include HCDR3, and the HCDR3 may include the following amino acid sequence: SEQ ID NO: 41.
  • the antibody heavy chain or a fragment thereof may include a heavy chain variable region VH, and the heavy chain variable region VH may include the following amino acid sequence: SEQ ID NO: 43.
  • the amino acid sequence of the antibody heavy chain or fragment thereof may be: SEQ ID NO: 48.
  • the amino acid sequence of LCDR1 in the antibody or antigen-binding fragment thereof described herein may include SEQ ID NO: 36 or its variant; the amino acid sequence of LCDR2 may include SEQ ID NO: 37 or its variant.
  • the amino acid sequence of LCDR3 may include SEQ ID NO: 38 or its variant; and the amino acid sequence of HCDR1 may include SEQ ID NO: 39 or its variant; the amino acid sequence of HCDR2 may include SEQ ID NO: 40 or its variant;
  • the amino acid sequence of HCDR3 may include SEQ ID NO: 41 or a variant thereof.
  • the antibody or antigen-binding fragment thereof may include the antibody SG701 or the antibody having the same LCDR1-3 and HCDR1-3 as it.
  • the light chain of the antibody or antigen-binding fragment thereof described herein may include a light chain variable region, and the amino acid sequence of the light chain variable region may include SEQ ID NO: 42 or a variant thereof ; And wherein the heavy chain may comprise a heavy chain variable region, the amino acid sequence of the heavy chain variable region may include SEQ ID NO: 43 or a variant thereof.
  • the antibody or antigen-binding fragment thereof may include the antibody SG701 or an antibody having the same light chain variable region and heavy chain variable region.
  • the antibody or antigen-binding fragment thereof described herein may comprise a light chain and a heavy chain, the light chain amino acid sequence of the light chain is shown in SEQ ID NO: 46 and the heavy chain amino acid sequence As shown in SEQ ID NO: 48.
  • the antibody or antigen-binding fragment thereof may include the antibody SG701 or the same light chain and heavy chain amino acid sequences as the antibody SG701.
  • the antibody described in this application may be SG701.
  • the amino acid sequence of LCDR1-3 of antibody SG701 is shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 42; the amino acid sequence of HCDR1-3 As shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 43; the amino acid sequence of the light chain is shown in SEQ ID NO: 46; heavy The amino acid sequence of the chain is shown in SEQ ID NO: 48.
  • the first binding domain may be located at the N-terminus of the second binding domain.
  • the C-terminus of the first binding domain may be indirectly connected to the N-terminus of the second binding domain through a linker.
  • the C-terminus of the first binding domain may also be directly (eg, in a box) connected to the N-terminus of the second binding domain.
  • the fusion protein may further include a linker, and the linker may be located at the C-terminus of the first binding domain and at the N-terminus of the second binding domain.
  • the C-terminus of the first binding domain may be connected to the N-terminus of the linker
  • the C-terminus of the linker may be connected to the N-terminus of the second binding domain.
  • a first binding domain, a linker, and a second binding domain may be sequentially included.
  • the linker may comprise the amino acid sequence shown in any one of SEQ ID NO: 73-74.
  • the fusion protein may comprise at least 2 (eg, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, At least 10, or more) the second binding domain.
  • each of the second binding domains may be located at the C-terminal of the first binding domain.
  • the two or more second binding domains may be directly or indirectly connected to the C-terminus of the first binding domain.
  • the fusion protein may include a first binding domain that specifically binds to CD38 antigen, and a second binding domain that specifically binds to CD47 protein, wherein the first binding domain may include an antibody that specifically binds to CD38 antigen Or the antigen-binding fragment or variant thereof, the second binding domain may comprise a mutant of human SIRP ⁇ variant 1, and the C-terminus of an antibody or antigen-binding fragment or variant that specifically binds CD38 antigen may be directly or indirectly linked to the human SIRP ⁇ variant N-terminal of the mutant of body 1.
  • the second binding domain may comprise at least 2 human SIRP ⁇ variant 1 mutants, and the N-terminal of the 2 human SIRP ⁇ variant 1 mutants are respectively connected to antibodies or antigen-binding fragments or variants that specifically bind to CD38 antigen C-terminal of the body.
  • the first binding domain of the fusion protein may include SG003, and the second binding domain may include 2 mutants M91 of SIRP ⁇ variant 1, and the sequence of linker 1 used is as SEQ As shown in ID NO: 73, the N ends of the two M91s are respectively connected to the C ends of the two heavy chains of SG003 through linker 1.
  • M91 can be connected to the C ends of the SG003 heavy chain to obtain the second most Peptide chain
  • the light chain of SG003 can be named as the first polypeptide chain.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SG3847L1 are shown in SEQ ID NO: 75 and SEQ ID NO: 11, respectively.
  • the first binding domain of the fusion protein may include SG003, and the second binding domain may include 2 mutants M91 of SIRP ⁇ variant 1, and the sequence of linker 2 used is as SEQ As shown in ID NO: 74, the N ends of the two M91s are respectively connected to the C ends of the two heavy chains of SG003 through the linker 2.
  • M91 is connected to the C ends of the SG003 heavy chain to obtain the second most Peptide chain
  • the light chain of SG003 can be named as the first polypeptide chain.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SG3847L2 are shown in SEQ ID NO: 76 and SEQ ID NO: 11, respectively.
  • the fusion protein may include a first binding domain that specifically binds to Trop2 antigen, and a second binding domain that specifically binds to CD47 protein, wherein the first binding domain may include an antibody that specifically binds to Trop2 antigen Or the antigen-binding fragment or variant thereof, the second binding domain may comprise a mutant of human SIRP ⁇ variant 1, and the C-terminus of an antibody or antigen-binding fragment or variant that specifically binds Trop2 antigen may be directly or indirectly linked to the human SIRP ⁇ variant N-terminal of the mutant of body 1.
  • the second binding domain may comprise at least 2 human SIRP ⁇ variant 1 mutants, and the N-terminus of 2 human SIRP ⁇ variant 1 mutants are respectively connected to antibodies or antigen-binding specifically binding to AXL antigen The C-terminus of the fragment or variant.
  • the first binding domain of the fusion protein may include antibody SG701
  • the second binding domain may include 2 mutants M91 of SIRP ⁇ variant 1
  • the sequence of the linker used is SEQ
  • ID NO: 74 the N-terminals of two M91s are connected to the C-terminals of the two heavy chains of SG701 through a linker respectively.
  • M91 is connected to the C-terminals of the SG701 heavy chain to obtain a second polypeptide Chain
  • the light chain of SG701 can be named as the first polypeptide chain.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGTrop247 are shown in SEQ ID NO: 78 and SEQ ID NO: 46, respectively.
  • the fusion protein may include a first binding domain that specifically binds to AXL antigen, and a second binding domain that specifically binds to CD47 protein, wherein the first binding domain may include an antibody that specifically binds to AXL antigen Or the antigen-binding fragment or variant thereof, the second binding domain may comprise a mutant of human SIRP ⁇ variant 1, and the C-terminus of an antibody or antigen-binding fragment or variant that specifically binds to AXL antigen may be directly or indirectly linked to human SIRP ⁇ N-terminal of the mutant of body 1.
  • the second binding domain may comprise at least 2 human SIRP ⁇ variant 1 mutants, and the N-terminus of 2 human SIRP ⁇ variant 1 mutants are respectively connected to antibodies or antigen-binding specifically binding to AXL antigen The C-terminus of the fragment or variant.
  • the first binding domain of the fusion protein may include antibody C6G12
  • the second binding domain may include two mutant M91 of SIRP ⁇ variant 1
  • the sequence of the linker used is SEQ
  • ID NO: 74 the N ends of the two M91s are connected to the C ends of the two heavy chains of C6G12 through a linker respectively.
  • M91 is connected to the C ends of the C6G12 heavy chain to obtain a second polypeptide Chain
  • the light chain of C6G12 can be named the first polypeptide chain.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGAXL47 are shown in SEQ ID NO: 77 and SEQ ID NO: 32, respectively.
  • each nucleic acid molecule of the one or more nucleic acid molecules may encode the entire antibody or antigen-binding fragment thereof, or may encode a portion thereof (eg, HCDR1-3, LCDR1-3, VL, VH , One or more of light chain or heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by: (i) amplified in vitro, such as polymerase chain reaction (PCR) amplification, (ii) cloned and recombinantly produced, (iii) purified , Such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • Recombinant DNA and molecular cloning techniques include by Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by TJSilhavy, ML Bennan and LWEnquist, Experiments with Genes Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), and Ausubel, FM, etc., Current Protocols, Molecular Biology, pub. By Greene Publishing Assoc. And Wiley-Interscience (1987) The techniques described.
  • the nucleic acids can be prepared from genomic DNA fragments, cDNA and RNA, and all these nucleic acids can be directly extracted from cells or recombinantly produced by various amplification methods including but not limited to PCR and RT-PCR.
  • Direct chemical synthesis of nucleic acids generally involves the sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of the growing nucleotide polymer chain, where each addition passes through the nucleophilic This is achieved by attacking the terminal 5'-hydroxyl group of the growing chain at the 3'-position of the added monomer, which is usually a phosphorus derivative, such as phosphotriester, phosphoramidite and the like. See, for example, Matteuci et al., Tet. Lett. 521: 719 (1980); US Patent No. 4,500,707 belonging to Caruthers et al .; and US Patent Nos.
  • the vector may be any linear nucleic acid, plasmid, phagemid, cosmid, RNA vector, viral vector, etc.
  • viral vectors can include retroviruses, adenoviruses, and adeno-associated viruses.
  • the vector is an expression vector, for example, a phage display vector.
  • the present application provides one or more vectors that contain the nucleic acid molecule.
  • the vector may contain one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is an adjustable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually contains 5 'untranscribed sequences and 5' and 3 'untranslated sequences involved in transcription and translation initiation, such as TATA box, Cap sequence, CAAT sequence, etc.
  • the 5 ' non-transcriptional expression control sequence may comprise a promoter region, and the promoter region may comprise a promoter sequence for transcriptional control of functionally linked nucleic acids.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoters, CMV promoters and their artificial hybrid promoters (such as CMV), wherein A certain part may be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, cosmid, virus, bacteriophage, or other vectors commonly used in, for example, genetic engineering.
  • the vector may be an expression vector.
  • the vector may also contain one or more selection marker genes, which after expression confer one or more phenotypic traits that can be used to select or otherwise identify the host cell carrying the vector.
  • suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.
  • the present application provides a cell, the cell comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, or the carrier.
  • the cell may be a host cell.
  • the cells may include many cell types such as prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 fruit fly cells or Sf9, or fibroblasts, CHO Animal cells such as cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • the vector can be stably or transiently introduced into the host cell by a variety of established techniques.
  • one method involves calcium chloride treatment in which a carrier is introduced by calcium precipitation.
  • Other salts such as calcium phosphate can also be used in a similar manner.
  • electroporation can be used (ie, current is applied to increase the permeability of cells to nucleic acids).
  • conversion methods include microinjection, DEAE dextran-mediated conversion, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes and dendrimers can also be used to transfect host cells.
  • An exemplary selection method includes subculturing a single cell to form a single colony, and then testing the expression of the desired protein product. Another method requires the selection of a host cell containing a heterologous sequence based on the phenotypic trait conferred by the expression of the selectable marker gene contained in the vector.
  • nucleic acid can be prepared from the resulting host cell, and a specific target sequence can be amplified by PCR using primers specific to the target sequence.
  • the amplified products are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, and then stained with ethidium bromide, SYBR Green solution, etc., or UV detection to detect DNA.
  • a nucleic acid probe specific for the target sequence can be used in the hybridization reaction.
  • the expression of a specific gene sequence can be determined by hybridization with PCR, Northern blotting, or detection of the corresponding mRNA by reverse transcription using immunoassay using antibodies that react with the encoded gene product.
  • immunoassays include, but are not limited to ELISA, radioimmunoassay, and sandwich immunoassay.
  • the introduction of various heterologous sequences of the present application into the host cell can be confirmed by the enzymatic activity of the enzyme (e.g., enzymatic marker) encoded by the heterologous sequence.
  • the enzyme can be determined by various methods known in the art. Generally, the enzymatic activity can be determined by the formation of the product or the conversion of the substrate of the enzymatic reaction under investigation. The reaction can be performed in vitro or in vivo.
  • the present application provides a method for preparing the fusion protein, which may include culturing the cell under conditions that enable expression of the fusion protein.
  • culturing the cell under conditions that enable expression of the fusion protein For example, it can be understood by those of ordinary skill in the art by using an appropriate culture medium, an appropriate temperature, and culture time.
  • the method may further include the step of isolating and / or purifying the fusion protein.
  • protein G-agarose or protein A-agarose can be used for affinity chromatography, and the fusion protein described in this application can also be purified and separated by gel electrophoresis and / or high performance liquid chromatography.
  • the present application provides an immunoconjugate comprising the fusion protein.
  • the immunoconjugate may be a fusion protein-drug conjugate (ADC), wherein the fusion protein described herein is conjugated to one or more therapeutic agents, including but not limited to cytotoxicity Agents, radiotoxic agents (eg, radioisotopes) and / or immunosuppressive agents (eg, any agent that kills cells by suppressing the immune response, etc.), etc.
  • the therapeutic agent may be a therapeutic agent capable of treating a tumor-related disease or disorder.
  • the conjugation can be performed by a peptide linker (eg, a cleavable linker) or other means, for example, the linker can be an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, or the like.
  • a peptide linker eg, a cleavable linker
  • the linker can be an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, or the like.
  • the present application provides a composition comprising the fusion protein, the immunoconjugate, or the nucleic acid molecule, and optionally a pharmaceutically acceptable excipient .
  • the pharmaceutically acceptable excipients may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or Nonionic surfactants, etc.
  • composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional technical means in the art, for example, according to Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing, Co., Easton, PA, 1995.
  • the composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, percutaneous administration
  • the drug may be administered through a subcutaneous depot.
  • the composition can be used to inhibit tumor growth.
  • the composition of the present application can inhibit or delay the development or progression of the disease, can reduce the size of the tumor (even substantially eliminate the tumor), and / or can reduce and / or stabilize the disease state.
  • the composition described in this application may be in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained-release preparations, solutions, suspensions, or for parenteral injection, such as Bacteria solution, suspension or emulsion, or for ointment or cream topical administration or rectal administration as suppository.
  • the composition may be in unit dosage forms suitable for single administration of precise doses.
  • the composition may further comprise conventional pharmaceutical carriers or excipients.
  • the composition may include other drugs or agents, carriers, adjuvants, and the like.
  • the composition described herein may contain a therapeutically effective amount of the fusion protein.
  • the therapeutically effective amount is a dose required to prevent and / or treat (at least partially treat) a condition or condition (eg, tumor) and / or any complications thereof in a subject suffering from or at risk of development.
  • the specific amount / concentration of the dose may vary according to the method of administration and the needs of the patient, and may be determined based on, for example, patient volume, viscosity, and / or body weight, and the like.
  • a suitable dose may be about 0.1 mg or 1 mg / kg / day to about 50 mg / kg / day; sometimes, the dose may be higher. It should be understood that those skilled in the art (for example, a doctor or pharmacist) can easily adjust these specific doses based on the specific patient, preparation, and / or disease status.
  • treatment or “treatment” or “remission” or “improvement” are used interchangeably in this application and refer to obtaining beneficial or desired results (including but not limited to therapeutic benefits and / or Preventive benefits).
  • therapeutic benefit generally refers to eradication or reduction of the severity of the underlying condition being treated.
  • therapeutic benefits by eradicating, reducing the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition, so that an improvement is observed in the subject (although the subject may still suffer from the underlying condition)
  • the composition may be administered to a subject at risk of developing a specific disease, or a subject reporting one or more physiological symptoms of the disease, even though the diagnosis of the disease may not have been performed.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the composition, or the use of the cell in the preparation of medicines , Where the drug can be used to treat tumors or autoimmune diseases.
  • the fusion protein, immunoconjugate, nucleic acid molecule, carrier, composition or cell described in this application can be used to treat the tumor or autoimmune disease.
  • the present application provides a method of treating a tumor or an autoimmune disease, the method comprising administering to the subject the fusion protein, immunoconjugate, nucleic acid molecule, carrier, composition, or cell described herein.
  • the present application provides a method of blocking the interaction of CD47 protein with SIRP ⁇ , which may include administration (eg, administration to a subject or cell or biological sample in need) of the application Fusion protein or composition.
  • the present application provides a method of inhibiting the growth and / or proliferation of a tumor or tumor cell, which may include contacting the fusion protein or composition described herein with the tumor or tumor cell.
  • the contact can occur in vitro.
  • the tumor may include non-solid tumors and solid tumors.
  • the tumor may include multiple myeloma, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma, germ cell tumor, sarcoma, epidermal tumor, placental tumor, Brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, prostate cancer, intestinal cancer, breast cancer, cervical cancer, ovarian cancer and testicular cancer.
  • the autoimmune disease may include chronic lymphatic thyroiditis, hyperthyroidism, insulin-dependent diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhagic nephritis Syndrome (goodpasture), pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebrospinal sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid joints Inflammation, scleroderma and polyarteritis nodosa.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the composition, or the cell, which can treat tumors or Autoimmune diseases.
  • the present application provides a method for blocking the interaction between CD47 protein and SIRP ⁇ , which may include administering an effective amount of the fusion protein, the immunoconjugate to a subject in need, The nucleic acid molecule, the carrier, the composition, or the cell.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cattle, sheep, goats, rabbits, mice, rats, or monkeys.
  • the present application provides a method for inhibiting the growth and / or proliferation of tumors or tumor cells, which may include administering an effective amount of the fusion protein to a subject in need, the immunoconjugation Substance, the nucleic acid molecule, the carrier, the composition, or the cell.
  • the amino acid sequence of LCDR1-3 of antibody SG003 is shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 7; the nucleotide encoding VL The sequence is shown in SEQ ID NO: 9; the amino acid sequences of HCDR1-3 of antibody SG003 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 8; the nucleotide sequence encoding VH is shown in SEQ ID NO: 10.
  • the amino acid sequence of the light chain of antibody SG003 is shown in SEQ ID NO: 11; the nucleotide sequence encoding it is shown in SEQ ID NO: 12.
  • the amino acid sequence of the heavy chain of antibody SG003 is shown in SEQ ID NO: 13; the nucleotide sequence encoding it is shown in SEQ ID NO: 14.
  • Select linker 1 (SEQ ID: NO 73), the constructed fusion protein is named SG3847L1, the SG3847L1 is composed of a first polypeptide chain and a second polypeptide chain, the first polypeptide chain is the light chain of SG003, and its amino acids
  • the sequence is shown in SEQ ID NO: 11
  • the second polypeptide chain is the heavy chain of SG003 and the polypeptide chain obtained by connecting M91 through linker 1
  • its amino acid sequence is shown in SEQ ID NO: 75.
  • the constructed fusion protein is named SG3847L2
  • the SG3847L2 is composed of a first polypeptide chain and a second polypeptide chain
  • the first polypeptide chain is the light chain of SG003, its amino acids
  • the sequence is shown in SEQ ID NO: 11
  • the second polypeptide chain is the heavy chain of SG003
  • the polypeptide chain obtained by connecting M91 through linker 2 and its amino acid sequence is shown in SEQ ID NO: 76.
  • the SG3847L1, SG3847L2 and SG003 antibodies have similar binding capacity to the target antigen CD38.
  • the EC50 values of SG3847L1, SG3847L2 and SG003 are 0.175 nM, 0.149 nM and 0.133 nM, respectively.
  • SG3847L1, SG3847L2 and SS002M91 have similar binding capacity to target antigen CD47.
  • the EC50 values of SG3847L1, SG3847L2 and SS002M91 are 0.114nM, 0.091nM and 0.072nM, respectively.
  • a CHO-S cell line engineered to express human CD38 or human CD47 was used to evaluate the fusion protein's ability to bind cell surface antigens. Collect cells in logarithmic growth phase, adjust cell density to 5 ⁇ 10 6 cells / mL, and pre-chill on ice. Normal saline with 2% FBS pre-chilled diluted SG003, SS002M91, negative control antibody (IgG1), SG3847L1 or SG3847L2 to different concentrations.
  • the fusion proteins SG3847L1, SG3847L2 and SG003 antibodies can simultaneously bind to the target antigens CD38 and CD47.
  • the EC50 values of SG3847L1 and SG3847L2 are 0.020 nM and 0.024 nM, respectively.
  • a CHO-S cell line engineered to express human CD38 was used to evaluate the ability of the fusion protein to simultaneously bind cell surface double antigens. Collect cells in logarithmic growth phase, adjust cell density to 5 ⁇ 10 6 cells / mL, and pre-chill on ice. Normal saline containing 2% FBS was diluted with SG003 antibody, SS002M91 fusion protein, negative control antibody (IgG1), SG3847L1 or SG3847L2. Take 100 ⁇ l of cells, add an equal volume of the aforementioned diluted antibody or fusion protein and CD47-FITC (Jiaxuan Biological), and react at 4 ° C in the dark for 30 min. After the end, the cells were washed twice.
  • Goat anti-human IgG Fc Secondary Antibody (PE-Goat anti-human IgG Fc Secondary Antibody, eBioscience), diluted with PE, was resuspended in 100 ⁇ L cells and reacted at 4 ° C in the dark for 30 min. After the reaction, the cells were washed twice. Resuspend the cells with 400 ⁇ l of 1% paraformaldehyde. Flow cytometry (BD Calibur) analyzes the ability of the fusion protein to bind to CD38 and CD47 on the cell surface simultaneously.
  • BD Calibur Flow cytometry
  • the fusion proteins SG3847L1 and SG3847L2 can simultaneously recognize the CD38 and CD47 antigens on the cell surface, and SG3847L2 has a higher ability to simultaneously recognize dual antigens.
  • LDH reaction mixture 100 ⁇ L / well in LDH detection kit (Cytotoxicity Detection Kit, Roche), 37 ° C React for 10 minutes.
  • the microplate reader measures OD492nm and OD650nm as the background value.
  • control 1 is ADCC buffer
  • control 2 is target cells + ADCC buffer
  • control 3 is target cells + lysate + ADCC buffer
  • control 4 is target cells + effector cells + ADCC buffer liquid.
  • Specific killing rate% ((Experimental group-Control 4) / (Control 3-Control 2)) ⁇ 100%.
  • the dose-effect curve adopts GraphPad Prism Version 5 for data analysis.
  • the fusion proteins SG3847L1 and SG3847L2 showed a better ADCC effect.
  • the EC50 values of SG003, SG3847L1 and SG3847L2 are 0.067nM, 0.014nM and 0.013nM, respectively.
  • SIRP ⁇ -His coated enzyme-linked plate 1 ⁇ g / ml, overnight at 4 °C; after washing with PBST, add 10% fetal bovine serum and block at 37 °C for 1 hour; use 10% fetal bovine blood to dilute SS002M91, SG3847L1 or SG3847L2, and add Biotin-Fc-CD47 to the sample to a final concentration of 2 ⁇ g / ml, pre-incubate at 37 ° C for 30 min as the primary antibody; after washing the enzyme-linked plate with PBST, add the primary antibody and incubate at 37 ° C for 1 hour; wash 5 times with PBST , Add horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiaxuan Bio), incubate at 37 °C for 30 minutes; wash 5 times with PBST, add 100 ⁇ l TMB (eBioscience) to each well, room temperature (20 ⁇ 5 °C) Place in the dark for
  • SG3847L1 and SG3847L2 can competitively block the binding of CD47 to its ligand SIRP ⁇ .
  • the IC50 value of SG3847L1 is 46.8nM
  • the IC50 value of SG3847L2 is 78.8nM
  • the IC50 value of SS002M91 is 45.7nM.
  • the anti-AXL antibody (C6G12) and anti-Trop2 (SG701) antibodies were selected as examples to study the influence of the biological activity of the fusion protein of different target antibodies.
  • the linker used is Connector 2.
  • the amino acid sequence of LCDR1-3 of antibody C6G12 is shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 28; the amino acid sequence of HCDR1-3 As shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 29; the amino acid sequence of the light chain is shown in SEQ ID NO: 32; heavy The amino acid sequence of the chain is shown in SEQ ID NO: 34.
  • the amino acid sequence of LCDR1-3 of antibody SG701 is shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39 respectively; the amino acid sequence of VL is shown in SEQ ID NO: 42; the amino acid sequence of HCDR1-3 As shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41 respectively; the amino acid sequence of VH is shown in SEQ ID NO: 43; the amino acid sequence of the light chain is shown in SEQ ID NO: 46; heavy The amino acid sequence of the chain is shown in SEQ ID NO: 48.
  • Fusion protein was prepared with anti-AXL antibody (C6G12), mutant M91 of SIRP ⁇ variant 1 and linker 2 (SEQ ID: NO 74), from C-terminal to C-terminal, C6G12, linker and two M91 were connected in sequence, The N-terminals of the two M91s were respectively connected to the C-terminals of the C6G12 heavy chain, and the resulting fusion protein was named SGAXL47.
  • SGAXL47 The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGAXL47 are shown in SEQ ID NO: 77 and SEQ ID NO: 32, respectively.
  • a fusion protein was prepared with anti-Trop antibody (SG701), mutant M91 of SIRP ⁇ variant 1 and linker 2 (SEQ ID: NO 74), and from the N-term to the C-terminus, SG701, linker and two M91 were connected in sequence The N-terminals of the two M91s were respectively connected to the C-terminal of the heavy chain of SG701, and the resulting fusion protein was named SGTrop247.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGTrop247 are shown in SEQ ID NO: 78 and SEQ ID NO: 46, respectively.
  • the expression genes of SGAXL47 and SGTrop247 fusion proteins were constructed into eukaryotic expression vectors. After transfection into CHO-S cells, the supernatant was harvested, and after protein affinity purification, the target protein was obtained and its biology was evaluated reasonably. active.
  • ELISA evaluated the binding activity of the corresponding fusion protein and related antigens.
  • SGAXL47 and C6G12 have similar binding capacity to AXL antigen.
  • SGTrop247 and SG701 have similar binding ability to Trop2 antigen. It means that the use of antibodies with different targets in the fusion protein does not affect the binding of the antibody to Trop2 antigen.
  • SGAXL47 and SS002M91 have similar binding ability to CD47 antigen.
  • SGTrop247 and SS002M91 have similar binding ability to CD47 antigen. This indicates that the use of antibodies with different targets in the fusion protein does not affect the binding of the antibody to its CD47 antigen.
  • ELISA analyzes the biological activity of the fusion protein while binding the double antigens.
  • Fig. 13A shows the binding of SGAXL47 and C6G12 to AXL and CD47 dual antigens respectively
  • Fig. 13B shows the binding of SGTrop247 and SS002M91 to Trop2 and CD47 dual antigens respectively.
  • the results in Figure 13 show that the fusion protein replaces different antibodies without affecting their simultaneous binding to the dual antigen.
  • Example 3 Fusion protein inhibits tumor activity in vivo
  • the tail vein of CB17SCID mice was inoculated with Raji-Luc cells to establish a tumor model, and the effect of fusion protein SG3847L1 on tumor activity was evaluated.
  • CB17SCID mice Female, 5-8 week old CB17SCID mice (purchased from Beijing Bioset Biotech Co., Ltd.) were selected for the experiment.
  • Raji-Luc cell is a stable cell line obtained by Beijing Biostar Biotech Co., Ltd. on the basis of Raji cell and transferred with luciferin reporter gene. After the Raji-Luc cells were recovered and cultured to the required number, the logarithmic growth cells were collected and suspended to a concentration of 2.5 ⁇ 10 7 cells / mL, and CB17SCID mice were inoculated through the tail vein at an amount of 0.2 mL / cell. 7. On the 7th day, observe the growth and weight of the tumor with a small animal imager.
  • mice On the 7th day, select 30 mice with moderate tumor imaging signals and randomly assign them to 5 groups, 6 in each group. Then start to administer the animal once.
  • Set saline control group experimental group (SG003, 100ug / kg), experimental group (SS002M91, 50ug / kg), co-administration group (SG003 + SS002M91, 100ug / kg + 50ug / kg), the experimental group (SG3847L1, 120ug / kg), observe the body weight and tumor growth of mice.
  • G1 physiological saline “G2SG003”, “G3SS002M91”, “G4SG003 + SS002M91” and “G5SG3847L1” in the figure represent “physiological saline group” and “SG003 antibody treatment group” "SS002M91 fusion protein treatment group”, “SG003 and SS002M91 combination treatment group” and “SG3847L1 fusion protein treatment group”, the median survival time of the saline group was 11 days, SG003 antibody treatment group and SS002M91 fusion protein treatment group median The survival period was 22 days and 21 days, respectively, indicating that SG003 and SS002M91 significantly prolonged the survival period of mice.
  • mice in the SG003 and SS002M91 combination treatment group and the SG3847L1 fusion protein treatment group showed death. It shows that compared with SG003 and SS002M91 alone treatment, SG003 and SS002M91 combination treatment group and SG3847L1 alone treatment group all prolong the survival time of mice.

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Abstract

提供的是一种融合蛋白，其包含特异性结合肿瘤相关抗原的第一结合域，以及特异性结合CD47蛋白的第二结合域；其中，所述第二结合域包含人SIRPα变体1的突变体。还提供了与所述融合蛋白相关的免疫缀合物、核酸分子、载体、组合物、细胞和制备方法，以及它们在制备药物中的用途。

Description

一种融合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种多特异性融合蛋白，还涉及其在治疗肿瘤和/或自免疫疾病中的用途。
背景技术
目前，在肿瘤治疗领域，存在施用靶向药物和免疫治疗两大方法。这两种治疗方法可能有相互作用，导致更强的细胞毒性作用，稳定而持续地缓解肿瘤。然而，靶向药物和免疫治疗之间的相互作用是非常复杂的，种类、剂量、顺序、剂型等各种因素都可能会影响联合治疗的整体抗肿瘤效果和毒性特征。
CD47蛋白是一种跨膜糖蛋白，属于免疫球蛋白超家族成员，除正常组织细胞表达CD47以外，许多肿瘤细胞过度表达CD47。肿瘤细胞表面的CD47与巨噬细胞表面的SIRPα相结合会阻止巨噬细胞对肿瘤细胞的吞噬，这被视为肿瘤逃避机体免疫监视的一种机制。阻断CD47蛋白和SIRPα的相互作用，可抑制肿瘤增长。
然而，现有的用于阻断CD47蛋白和SIRPα相互作用的试剂识别活性有限，其与CD47蛋白的亲和力往往不足，对肿瘤的抑制能力有限。同时，现有靶向CD47的抗体类药物存在引起贫血反应或者血小板减少的副作用。亟待获得同时特异性靶向CD47蛋白和相关肿瘤抗原的有效疗法。
发明内容
本申请提供了一种融合蛋白，其包含特异性结合肿瘤相关抗原的第一结合域和特异性结合CD47蛋白的第二结合域。本申请还提供包含该融合蛋白的免疫缀合物；编码该融合蛋白的核酸分子；能够包含和/或表达该融合蛋白的载体、组合物和细胞；以及制备所述融合蛋白的方法。本申请的融合蛋白、免疫缀合物、核酸分子、载体、组合物和细胞具有以下一种或多种性质：1)能够同时特异性结合CD47蛋白和肿瘤相关抗原；2)能够特异性阻断CD47蛋白与SIRPα的相互作用；3)能够有效抑制肿瘤或肿瘤细胞的生长和/或增殖。
一方面，本申请提供了一种融合蛋白，其包含：特异性结合肿瘤相关抗原的第一结合域；以及特异性结合CD47蛋白的第二结合域；其中，所述第二结合域包含人SIRPα变体1的突变体，所述突变体与SEQ ID NO:50所示的序列相比，在第33位至第149位中的一个或多个位置包含氨基酸残基的取代、缺失或添加。
在某些实施方式中，所述突变体在选自下组的一个或多个氨基酸残基处包含氨基酸取代：R22、I29、I61、V63、E77、Q82、K83、E84、V93、D95、D96、K98、N100、R107、G109和V132。
在某些实施方式中，所述突变体在选自下组的氨基酸残基处包含氨基酸取代：(1)I61、V63、E77、E84、V93、L96、K98、N100和V132；(2)I61、E77、Q82、K83和E84；(3)I61、V63、K83、E84和V132；(4)I61、E77、E84、R107和V132；(5)I61、V63、E77、K83、E84和N100；(6)I61、E77、Q82、K83、E84和R107；(7)I61、E77、Q82、E84、V93、L96、N100、R107、G109和V132；(8)I61、E77、Q82、K83、E84和V132；(9)I61；(10)I61、D95、L96、G109和V132；(11)I61、D95、L96、K98、G109和V132；(12)I61、E77、E84、V93、R107和V132；(13)E77、L96、N100、G109和V132；(14)I61、V63、Q82、E84、D95、L96、N100和V132；(15)I61、E77、Q82、K83、E84、V93、D95、L96、K98、N100和V132；(16)I61、E77、Q82、K83、E84和V93；(17)I61、V63、E77、K83、E84、D95、L96、K98和N100；(18)I61、V63、E77、K83、D95、L96、K98、N100和G109；(19)I61、E77、Q82、E84、V93、D95、L96、K98和N100；和，(20)I61、V63、E77、Q82和E84。
在某些实施方式中，所述突变体包含选自下组的一个或多个氨基酸取代：R22C、I29L、I61L/V/F、V63I、E77I/N/Q/K/H/M/R/N/V/L、Q82S/R/G/N、K83R、E84K/H/D/R/G、V93L/A、D95H/R/E、D96S/T、K98R、N100G/K/D/E、R107N/S、G109R/H和V132L/R/I/S。
在某些实施方式中，所述突变体包含选自下组的氨基酸取代：(1)I61L、V63I、E77I、E84K、V93L、L96S、K98R、N100G和V132L；(2)I61V、E77N、Q82S、K83R和E84H；(3)I61F、V63I、K83R、E84K和V132I；(4)I61L、E77Q、E84D、R107N和V132I；(5)I61L、V63I、E77K、K83R、E84D和N100G；(6)I61V、E77H、Q82R、K83R、E84H和R107S；(7)I61L、E77I、Q82G、E84R、V93L、L96T、N100G、R107S、G109R和V132R；(8)I61L、E77M、Q82G、K83R、E84D和V132L；(9)I61L；(10)I61F、D95H、L96S、G109H和V132S；(11)I61F、D95H、L96S、K98R、G109H和V132S；(12)I61L、E77Q、E84D、V93A、R107N和V132I；(13)E77K、L96S、N100K、G109H和V132L；(14)I61L、V63I、Q82G、E84G、D95R、L96S、N100D和V132I；(15)I61L、E77R、Q82N、K83R、E84G、V93L、D95E、L96T、K98R、N100D和V132L；(16)I61V、E77N、Q82S、K83R、E84H和V93A；(17)I61V、V63I、E77V、K83R、E84D、D95E、L96T、K98R和N100E；(18)I61L、V63I、E77V、K83R、D95E、L96S、K98R、N100D和G109R；(19)I61V、E77L、Q82G、E84G、V93L、D95E、L96T、K98R和N100G；和，(20)I61L、V63I、E77N、 Q82G和E84G。
在某些实施方式中，所述突变体包含如SEQ ID NO:51-70中任一项所示的氨基酸序列。
在某些实施方式中，所述第一结合域包含抗体或其抗原结合片段或变体。在某些实施方式中，所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab')2，F(ab)2，dAb，分离的互补决定区CDR，Fv和scFv。
在某些实施方式中，所述抗体或其抗原结合片段的所述变体选自下组：
a)在所述抗体或所述其抗原结合片段中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽；和
b)与所述抗体或所述其抗原结合片段具有至少90％序列同源性的蛋白质或多肽。
在某些实施方式中，所述肿瘤相关抗原包括与非实体瘤和/或实体瘤相关的肿瘤相关抗原。在某些实施方式中，所述肿瘤相关抗原选自以下组：CD38、AXL和Trop2。
在某些实施方式中，所述第一结合域包含CD38抗体或其抗原结合片段或变体。在某些实施方式中，所述CD38为人CD38。
在某些实施方式中，所述抗体包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6。在某些实施方式中，所述抗体重链或其片段包含重链可变区VH，且所述重链可变区VH包含选自下组的氨基酸序列：SEQ ID NO:8。在某些实施方式中，所述抗体重链或其片段包含重链恒定区，且所述重链恒定区包含IgG。在某些实施方式中，所述IgG选自下组：IgG1和IgG4。在某些实施方式中，所述抗体重链包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:19和SEQ ID NO:21。
在某些实施方式中，所述抗体包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3。在某些实施方式中，所述抗体轻链或其片段包含轻链可变区VL，且所述轻链可变区VL包含选自下组的氨基酸序列：SEQ ID NO:7。在某些实施方式中，所述抗体轻链或其片段包含轻链恒定区，其所述轻链恒定区包含Igκ。在某些实施方式中，所述抗体轻链包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:18和SEQ ID NO:20。
在某些实施方式中，所述第一结合域包含AXL抗体或其抗原结合片段或变体。在某些实施方式中，所述AXL为人AXL。
在某些实施方式中，所述抗体包含抗体重链或其片段，所述抗体重链或其片段包含 HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27。在某些实施方式中，所述抗体重链或其片段包含重链可变区VH，且所述重链可变区VH包含选自下组的氨基酸序列：SEQ ID NO:29。在某些实施方式中，所述抗体重链或其片段包含重链恒定区，且所述重链恒定区包含IgG。在某些实施方式中，所述IgG选自下组：IgG1和IgG4。在某些实施方式中，所述抗体重链包含选自下组的氨基酸序列：SEQ ID NO:34。
在某些实施方式中，所述抗体包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24。在某些实施方式中，所述抗体轻链或其片段包含轻链可变区VL，且所述轻链可变区VL包含选自下组的氨基酸序列：SEQ ID NO:28。在某些实施方式中，所述抗体轻链或其片段包含轻链恒定区，其所述轻链恒定区包含Igκ。在某些实施方式中，所述抗体轻链包含选自下组的氨基酸序列：SEQ ID NO:32。
在某些实施方式中，所述第一结合域包含Trop2抗体或其抗原结合片段或变体。在某些实施方式中，所述Trop2为人Trop2。
在某些实施方式中，所述抗体包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41。在某些实施方式中，所述抗体重链或其片段包含重链可变区VH，且所述重链可变区VH包含选自下组的氨基酸序列：SEQ ID NO:43。在某些实施方式中，所述抗体重链或其片段包含重链恒定区，且所述重链恒定区包含IgG。在某些实施方式中，所述IgG选自下组：IgG1和IgG4。在某些实施方式中，所述抗体重链包含选自下组的氨基酸序列：SEQ ID NO:48。在某些实施方式中，所述抗体包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38。在某些实施方式中，所述抗体轻链或其片段包含轻链可变区VL，且所述轻链可变区VL包含选自下组的氨基酸序列：SEQ ID NO:42。在某些实施方式中，所述抗体轻链或其片段包含轻链恒定区，其所述轻链恒定区包含Igκ。在某些实施方式中，所述抗体轻链包含选自下组的氨基酸序列：SEQ ID NO:46。
在某些实施方式中，所述第一结合域位于所述第二结合域的N端。在某些实施方式中，所述融合蛋白还包含连接子，所述连接子位于所述第一结合域的C端且位于所述第二结合域的N端。在某些实施方式中，所述连接子包含如SEQ ID NO:73-74中任一项所示的氨基酸序列。
在某些实施方式中，所述的融合蛋白包含至少2个所述第二结合域。在某些实施方式中， 所述每个所述第二结合域分别位于所述第一结合域的C端。
另一方面，本申请提供了一种免疫缀合物，其包含所述的融合蛋白。
另一方面，本申请提供了一个或多个分离的核酸分子，其编码所述的融合蛋白或者所述的免疫缀合物。
另一方面，本申请提供了一个或多个载体，其包含所述的核酸分子。
另一方面，本申请提供了一种组合物，其包含所述的融合蛋白，所述的免疫缀合物，或所述的核酸分子，以及任选地药学上可接受的赋形剂。
另一方面，本申请提供了一种细胞，其包含所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，或所述的载体。
另一方面，本申请提供了制备所述的融合蛋白的方法，其包括在使得所述融合蛋白能够表达的条件下培养所述的细胞。
另一方面，本申请提供了所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞在制备药物中的用途，其中所述药物用于治疗肿瘤或自免疫疾病。
在某些实施方式中，所述肿瘤包括非实体瘤和实体瘤。
在某些实施方式中，所述肿瘤包括多发性骨髓瘤、白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤、生殖细胞瘤、肉瘤、见皮瘤、胎盘瘤、脑癌、骨癌、皮肤癌、鼻咽癌、肺癌、口腔癌、食道癌、胃癌、肝癌、胰腺癌、前列腺癌、肠癌、乳腺癌、宫颈癌、卵巢癌和睾丸癌。
在某些实施方式中，所述自免疫疾病包括慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征(goodpasture syndrome)、寻常天皰疮、类天皰疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症、急性特发性多神经炎、系统性红斑狼疮、类风湿性关节炎、硬皮病和结节性多动脉炎。
另一方面，本申请提供了所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞，其治疗肿瘤或自免疫疾病。
另一方面，本申请提供了一种阻断CD47蛋白与SIRPα相互作用的方法，其包括向有需要的受试者施用有效量的所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞。
另一方面，本申请提供了一种抑制肿瘤或肿瘤细胞生长和/或增殖的方法，其包括向有需要的受试者施用有效量的所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载 体，所述的组合物，或所述的细胞。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述融合蛋白的结构示例。
图2和3显示的是本申请所述融合蛋白的生物学活性。
图4A和4B显示的是本申请所述融合蛋白的生物学活性。
图5显示的是检测本申请所述融合蛋白与CD38和CD47之间相互作用的方法原理示意图。
图6显示的是本申请所述融合蛋白的生物学活性。
图7显示的是检测本申请所述融合蛋白与CD38和CD47之间相互作用的方法原理示意图。
图8和9显示的是本申请所述融合蛋白的生物学活性。
图10显示的是本申请所述融合蛋白对CD47蛋白与SIRPα相互作用的阻断。
图11A-11B、图12A-12B以及图13A-13B显示的是本申请的融合蛋白与相应抗原的结合活性。
图14显示不同治疗组小鼠的肿瘤成像结果。
图15显示不同治疗组小鼠的肿瘤平均荧光强度。
图16显示不同治疗组小鼠的生存率。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“融合蛋白”通常指由两个或更多个蛋白或多肽融合得到的蛋白。融 合蛋白可通过重组DNA技术人工制备。例如，编码所述两个或更多个蛋白或多肽的基因或核酸分子可彼此连接而形成融合基因或融合的核酸分子，该融合基因或融合的核酸分子可编码所述融合蛋白。所述融合基因的翻译可以产生单一多肽，其可以具有融合前的所述两个或更多个蛋白或多肽中至少一个、甚至每一个的性质。
在本发明中，术语“特异性结合”通常是指两分子间的非随机结合反应，如抗体和产生该抗体的抗原间的反应。一个某抗原特异性抗体是指以亲和力(KD)≤10 -5M(如10 -6M、10 - 7M、10 -8M、10 -9M、10 -10M等)结合该抗原，其中KD指解离率与结合率的比值(koff/kon)，其可以采用本领域技术人员熟悉的方法进行测定。
在本申请中，术语“结合域”通常是可以特异性结合和/或识别靶标(例如抗原)上的特定表位的结构域。在本申请中，术语“结构域”通常是指蛋白质亚基结构中明显分开的紧密球状结构区域。例如，多肽链首先可以是在某些区域相邻的氨基酸残基形成有规则的二级结构，然后，又可以由相邻的二级结构片段组装在一起形成超二级结构，在此基础上多肽链可以折叠成近似于球状的三级结构。对于较大的蛋白质分子或亚基，多肽链往往可以由两个或多个在空间上可明显区分的、相对独立的区域性结构缔合而成三级结构，这种相对独立的区域性结构可以称为结构域。
在本申请中，术语“第一结合域”和“第二结合域”中的所述“第一”、“第二”可以只是为了在描述上进行区分。
在本申请中，术语“CD47蛋白”通常是指整联蛋白相关蛋白(IAP)，其为一种属于免疫球蛋白超家族的多次跨膜受体。例如，CD47蛋白可与膜整合素(membrane integrins)结合，并与其配体凝血栓蛋白-1(thrombospondin-1,TSP-1)和信号调节蛋白α(signal-regulatory protein alpha,SIRPα)结合。CD47蛋白广泛表达于细胞膜表面。在本申请中，所述CD47蛋白可包括人CD47的任何变体、同种型和物种同系物。人CD47蛋白的氨基酸序列在GenBank中作为CEJ95640.1列出。所述CD47蛋白可以由细胞天然表达或在用CD47基因转染的细胞上表达。
在本申请中，术语“SIRPα”通常是指来自SIRP家族的调节性膜糖蛋白，其可作为CD47蛋白的配体。在本申请中，所述SIRPα可包括人SIRPα。例如，SIRPα变体1和SIRPα变体2。所述SIRPα变体2与所述SIRPα变体1有13个氨基酸不同，并且其氨基酸序列在GenBank中作为CAA71403.1列出。在本申请中，术语“SIRPα变体1”通常是指氨基酸序列作为NCBI RefSeq NP_542970.1列出(残基31-504构成成熟型)的SIRPα蛋白，此时SIRPα变体1的氨基酸序列如SEQ ID NO:50所示。
在本申请中，术语“变体”通常是指与不具备任何突变/修饰的蛋白质具有序列同源性的蛋 白质性分子，其保留至少一部分生物活性蛋白的治疗和/或生物活性。例如，变体蛋白质相较于参照生物活性蛋白可共享至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的氨基酸序列同一性。在一些实施方案中，“变体”可以包括经有意地修饰(例如通过定点诱变、编码基因的合成、插入或偶然通过突变)的蛋白质。
术语“抗体”通常是指包含一个或多个基本上由免疫球蛋白基因或免疫球蛋白基因片段编码的多肽的蛋白质。例如，免疫球蛋白基因可以包括κ、λ、α、γ、δ、ε和μ恒定区基因，以及无数的免疫球蛋白可变区基因。例如，轻链可被分类为κ或λ，其可以分别定义免疫球蛋白类型：Igκ和Igλ。重链可被分类为γ、μ、α、δ或ε，其依次分别定义免疫球蛋白类别：IgG、IgM、IgA、IgD和IgE。例如，抗体可具有包含四聚体的结构单元，每个四聚体可由两对相同的多肽链组成，每对具有一条“轻”链(约25kD)和一条“重链”(约50-70kD)，每个成员的N末端可以界定约100至110个或更多个氨基酸的可变区，其主要负责抗原识别。例如，术语“轻链可变区(VL)”和“重链可变区(VH)”通常分别指轻链和重链的可变区区域。抗体可作为完整免疫球蛋白存在或作为通过用各种肽酶消化或从头表达产生的许多充分表征的片段存在。
在本发明中，术语“抗原结合片段”通常是指全长抗体的一个或多个部分，所述部分基本上保持结合抗体所结合的相同抗原(例如，CD38)的能力，能够与全长抗体竞争对抗原的特异性结合。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，并且将其全文通过引用并入本申请。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab'、F(ab')2、(Fab)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体获得抗体的抗原结合片段，并且以与对于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。例如，胃蛋白酶可以消化铰链区中二硫键以下的抗体以产生F(ab')2。
在本申请中，术语“Fab”通常指由VL、VH、CL和CH1结构域组成的抗体片段。
在本申请中，术语“Fab'”通常是指与Fab片段相比在CH1结构域的羧基末端具有几个额外的残基的抗体片段。例如，Fab'可包括来自抗体铰链区的一个或多个半胱氨酸。
在本申请中，术语“F(ab)2”通常是指由半胱氨酸相连接的成对的Fab片段所得到的抗原结合片段。
在本申请中，术语“dAb片段”通常是指由VH结构域组成的抗体片段(Ward等人,Nature  341:544-546(1989))。
在本申请中，术语“互补决定区CDR”通常是指轻链可变区(VL)与重链可变区(VH)的3个高变区(HVR)，该部位因在空间结构上可与抗原决定簇形成精密的互补，故高变区又称互补性决定区。
在本申请中，术语“Fv片段”通常是指由抗体的单臂的VL和VH结构域组成的抗体片段。
在本申请中，术语“scFv”通常是指是由抗体重链可变区和轻链可变区通过短肽连接子(linker)连接而成的分子，又称为单链抗体。
在本申请中，术语“单克隆抗体”通常是指一群基本同源的抗体，包含于该群的各个抗体除了可能的以微量存在的天然发生的突变之外可以是相同的。单克隆抗体是高度特异性的，直接针对单个抗原性位点。此外，与包括针对不同决定簇(表位)的不同抗体的多克隆抗体制备物相反，每个单克隆抗体针对抗原上的单一决定簇修饰语"单克隆"不是被解释为需要通过任何特殊方法产生抗体。例如，所述单克隆抗体可以通过杂交瘤技术制备或者通过使用重组DNA方法在细菌、真核动物或植物细胞中产生单克隆抗体，也可以得自噬菌体抗体文库，使用例如Clackson et al.,Nature,352:624-628(1991)和Marks et al.,Mol.Biol.，222:581-597(1991)所述的技术进行。
在本申请中，术语“嵌合抗体”通常是指这样的抗体，其中每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别，而该链的其余区段则与另一物种中的相应序列同源。例如，轻链和重链的可变区均来自一个动物物种(如小鼠、大鼠等)的抗体的可变区，而恒定部分则与来自另一物种(如人)的抗体序列同源。例如，为获得嵌合抗体，可利用非人源的B细胞或杂交瘤细胞产生可变区，而与其组合的恒定区则来自人。所述可变区具有易于制备的优点，并且其特异性不受与其组合的恒定区的来源的影响。同时，由于嵌合抗体的恒定区可来源于人类，因此嵌合抗体在注射时抗体引发免疫应答的可能性会低于使用恒定区为非人来源的抗体。
在本申请中，术语“人源化抗体”通常是指将衍生自非人物种(例如小鼠或大鼠)的抗体、免疫球蛋白结合蛋白和多肽对人体的免疫原性降低，同时仍保留原始抗体的抗原结合特性的改造抗体。例如，可以使用遗传工程技术制备人源化抗体，可以使用CDR移植(Jones et al.,Nature 321:522(1986))及其变体；包括“重塑”(reshaping)，(Verhoeyen,et al.,1988Science 239:1534-1536；Riechmann,et al.,1988 Nature 332:323-337；Tempest,et al.,Bio/Technol 1991 9:266-271)，“高度加成”(hyperchimerization)，(Queen,et al.,1989 Proc Natl Acad Sci USA 86:10029-10033；Co,et al.,1991 Proc Natl Acad Sci USA 88:2869-2873；Co,et al.,1992 J Immunol 148:1149-1154)和“贴面”(veneering)，(Mark,et al.,“Derivation of therapeutically  active humanized and veneered anti-CD18 antibodies.”In:Metcalf B W,Dalton B J,eds.Cellular adhesion:molecular definition to therapeutic potential.New York:Plenum Press,1994:291-312)等技术手段，对非人源的结合域进行人源化。如果其他区域，例如铰链区和恒定区结构域也源自非人来源，则这些区域也可以被人源化。
在本申请中，术语“全人源抗体”通常是指由基因工程改造的抗体基因缺失动物中表达编码人类抗体的基因得到的抗体。例如，可以将人类抗体基因通过转基因或转染色体技术，将编码人类抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达的人类抗体
如本申请中所用，术语“序列同源性”通常是指两个或多个多核苷酸序列之间或两个或多个多肽序列之间的序列相似性或可交换性。当使用程序(例如Emboss Needle或BestFit)来测定两个不同氨基酸序列之间的序列同一性、相似性或同源性时，可以使用默认设置，或者可以选择合适的评分矩阵(诸如blosum45或blosum80)来最优化同一性、相似性或同源性得分。在一些实施方案中，同源的多核苷酸是在严格条件下杂交的那些序列，并且相较于那些序列具有至少60％，至少65％，至少70％，至少80％，至少90％，至少95％，至少97％，至少98％，至少99％，甚至100％的序列同一性。当对准具有相当长度的序列时，同源的多肽具有至少80％，或至少90％，或至少95％，或至少97％，或至少98％的序列同一性，或具有至少99％的序列同一性。
在本申请中，术语“CD38”、“CD38蛋白”和“CD38抗原”在本申请中可互换使用，并且包括人CD38的任何变体、同种型和物种同系物，其可以由细胞天然表达或在用CD38基因转染的细胞上表达。本领域公认的CD38的同义词包括ADP核糖基环化酶1、cADPr水解酶1、Cd38-rs1、环状ADP-核糖水解酶1、I-19和NIM-R5抗原。CD38可以在骨髓瘤细胞表面高度表达。
在本申请中，术语“AXL”、“AXL蛋白”和“AXL抗原”在本申请中可以互换使用，通常是指属于酪氨酸激酶家族的一种蛋白，该家族可以由共同的配体Gas6。在本申请中，，AXL的高表达与肿瘤形成可以由较大的关联，AXL可以当作癌细胞恶化的标识物。在本申请中，所述AXL可以为人AXL，其氨基酸序列在GenBank中作为AAH32229列出。
在本申请中，术语“Trop2”通常是指肿瘤相关钙信号转导2(tumor-associated calcium signal transducer 2)，可以被称为TACSTD2、EGP-1、GA733-1或GP50等。Trop2在细胞中可以定位于细胞膜，其阳性染色着色部位可主要在细胞膜上。在正常组织中，Trop2的表达量很低，但在肿瘤细胞中过表达，因此可以作为肿瘤治疗的靶点。在本申请中，所述Trop2可以为人Trop2，其氨基酸序列在GenBank中作为NP_002344列出。
通常情况下，在多肽链中，氨基与多肽链中的另一个羧基相连可以使其成为一个链，但是在蛋白质的两个末端，分别剩余没有成肽键的氨基酸残基，分别是携带游离的氨基的多肽链末端和携带羧基的多肽链末端。在本申请中，术语“N端”通常是指氨基酸残基携带游离的氨基的多肽链的末端。在本申请中，术语“C端”通常是指氨基酸残基携带游离的羧基的多肽链的末端。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“免疫缀合物”通常是指缀合了一个或多个异源分子(包括但不限于细胞毒素)的具有免疫功能的多肽分子。在本申请中，“缀合”和“连接”、“融合”在本申请中可以互换使用，并且通常是指将两个或更多个化学元素、序列或组分连接在一起，例如通过包括化学缀合或重组手段。所述异源分子可以是细胞毒素、化疗药物等。例如，将本申请所述的融合蛋白缀合一个或多个异源分子(例如，细胞毒素)可以得到所述的免疫缀合物。
在本发明中，术语“载体”指的是，可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“肿瘤”通常是指哺乳动物中的机体(例如，细胞或其组成部分)在各种致瘤因子作用下，局部组织细胞增生所形成的赘生物。在本申请中，肿瘤可以包括实体瘤和非实体瘤。实体瘤可以包括神经胶质瘤、生殖细胞瘤、肉瘤、见皮瘤、胎盘瘤、脑癌、骨癌、皮肤癌、鼻咽癌、肺癌、口腔癌、食道癌、胃癌、肝癌、胰腺癌、前列腺癌、肠癌、乳腺癌、宫颈癌、卵巢癌和睾丸癌。在申请中，非实体瘤可以包括多发性骨髓瘤、白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤。
在本申请中，术语“淋巴瘤”通常是指一种淋巴系统的恶性肿瘤。淋巴瘤的发生可以是由于淋巴结细胞或淋巴细胞开始无法控制地繁殖，并产生具有异常能力，可以侵入整个身体其他组织的癌细胞。患者体征和症状可以包括淋巴结肿大、发热、盗汗、消瘦或瘙痒，并持 续感到疲倦。淋巴瘤有多种亚型，其中两种主要的类型可以为霍奇金淋巴瘤和非霍奇金淋巴瘤。在本申请中，术语“霍奇金淋巴瘤”(Hodgkin's lymphoma，HL)通常是指一种由淋巴细胞类白细胞产生的一种淋巴瘤。约有一半的霍奇金淋巴瘤病例是由Epstein-Barr病毒引起的。在本申请中，术语“非霍奇金淋巴瘤”(Non-Hodgkin lymphoma，NHL)通常是指除了霍奇金淋巴瘤外其他类型的淋巴瘤。
在本申请中，术语“白血病”通常是指一种造血系统恶性增殖性疾病，通常指因白血病细胞大量增殖、累积所引起的一类疾病。克隆性白血病细胞因为增殖失控、分化障碍、凋亡受阻等机制在骨髓和其他造血组织中大量增殖累积，并浸润其他非造血组织和器官，同时抑制正常造血功能。临床可见不同程度的贫血、出血、感染发热以及肝、脾、淋巴结肿大和骨骼疼痛。
在本申请中，术语“多发性骨髓瘤”通常是指一种浆细胞不正常增生致使侵犯骨髓的一种恶性肿瘤。多发性骨髓瘤导致癌细胞可以在骨髓中聚集，使得健康的血细胞被排除。癌细胞产生可导致并发症的异常蛋白质而不是正常的抗体。多发性骨髓瘤的发病率可以在老年人群中增加。其化学治疗方案细胞增殖速率低和药物抗药性强，限制了其化疗效果。超过90％的多发性骨髓瘤患者有化疗抗药性。
在本申请中，术语“Raji细胞”通常是指能产生Epstein-Barr病毒株的连续的人类细胞系。病毒将转化脐带淋巴细胞并且在Raji细胞中诱导早期抗原。Raji细胞可以被广泛用作转染宿主，也可以被用于了解造血细胞和其他细胞恶性肿瘤。此外，Raji细胞具有和表达某些补体成分的几种受体以及免疫球蛋白G的Fc受体，因而还可以被用于检测免疫复合物。
在本申请中，术语“自免疫疾病”通常是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病。自免疫疾病可以分为两大类，即器官特异性自身免疫病和系统性自身免疫病。器官特异性自身免疫病可以是指组织器官的病理损害和功能障碍仅限于抗体或致敏淋巴细胞所针对的某一器官，可包括慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征(goodpasture syndrome)、寻常天皰疮、类天皰疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症和急性特发性多神经炎。系统性自身免疫病可以是由于抗原抗体复合物广泛沉积于血管壁等原因导致全身的多器官损害，可以是由于免疫损伤导致血管壁及间质的纤维素样坏死性炎及随后产生多器官的胶原纤维增生所致，系统性自身免疫病可以包括系统性红斑狼疮、类风湿性关节炎、硬皮病和结节性多动脉炎。
在本申请中，术语“ADCC”是抗体依赖性细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity,ADCC)，通常是指是指具有杀伤活性的细胞通过其表面表达的Fc受体 (FcR)识别包被于靶抗原上的Fc段，由此免疫系统的效应细胞主动溶解其膜表面抗原已被特异性抗体结合的靶细胞。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
融合蛋白
一方面，本申请提供一种融合蛋白，所述融合蛋白可包含第一结合域以及第二结合域。所述第一结合域可以特异性结合肿瘤相关抗原；所述第二结合域可以特异性结合CD47蛋白，所述第二结合域可以包含人SIRPα变体1的突变体，所述突变体与SEQ ID NO:50所示的序列相比，在第33位至第149位中的一个或多个(例如，1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个或更多个)位置包含氨基酸残基的取代、缺失或添加。本申请所述的融合蛋白能够同时特异性结合肿瘤相关抗原和CD47蛋白，从而起到治疗肿瘤和/或自免疫疾病的作用。
在本申请中，术语“第一结合域”通常是指可以特异性结合肿瘤相关抗原的结构域。术语“第二结合域”通常是指可以特异性结合CD47蛋白的结构域。
在本申请中，所述肿瘤相关抗原可以包括与非实体瘤和/或实体瘤相关的肿瘤相关抗原。例如，所述肿瘤相关抗原可以选自以下组：CD38、AXL和Trop2。
特异性结合CD47的第二结合域
在本申请中，所述突变体(例如特异性结合CD47蛋白的人SIRPα变体1的突变体)在选自下组的一个或多个(例如，1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个或更多个)氨基酸残基处包含氨基酸取代：R22、I29、I61、V63、E77、Q82、K83、E84、V93、D95、D96、K98、N100、R107、G109和V132。
在本申请中，所述氨基酸取代中氨基酸残基的位置是以SEQ ID NO:50所示氨基酸序列为基准确定的残基编号。
在本申请中，“氨基酸取代Xn”是指在相应于SEQ ID NO:50所示氨基酸序列中第n位的残基X处发生氨基酸取代，其中n为正整数，X为任意氨基酸残基的缩写。例如，“氨基酸取代I61”表示在相应于SEQ ID NO:50所示氨基酸序列中第61位的残基I处发生氨基酸取代。
本申请中，所述的氨基酸取代可以为非保守取代。所述非保守取代可包括以非保守的形 式改变目标蛋白或多肽中的氨基酸残基，例如将具有某种侧链大小或某种特性(例如，亲水性)的氨基酸残基变为具有不同侧链大小或不同特性(例如，疏水性)的氨基酸残基。
本申请中，所述的氨基酸取代也可以为保守取代。所述保守取代可包括以保守的形式改变目标蛋白或多肽中的氨基酸残基，例如将具有某种侧链大小或某种特性(例如，亲水性)的氨基酸残基变为具有相同或相似侧链大小或者相同或相似特性(例如，仍为亲水性)的氨基酸残基。这样的保守取代通常不会对所产生的蛋白质的结构或功能带来很大影响。在本申请中，作为所述融合蛋白或其片段的氨基酸序列变体可包括不显著改变蛋白质结构或其功能(例如，阻断CD47与特异性结合CD47蛋白的人SIRPα变体1的突变体)的保守氨基酸取代。
作为示例，下述各组中每组内各氨基酸间的相互取代在本申请中可被认为是保守取代：具有非极性侧链的氨基酸组：丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、色氨酸和甲硫氨酸。不带电荷、具有极性侧链的氨基酸组：甘氨酸、丝氨酸、苏氨酸，半胱氨酸，酪氨酸，天冬酰胺和谷氨酰胺。带负电荷、具有极性侧链的氨基酸组：天冬氨酸和谷氨酸。带正电荷的碱性氨基酸：赖氨酸、精氨酸和组氨酸。带苯基的氨基酸：苯丙氨酸、色氨酸和酪氨酸。
在本申请中，所述突变体可以在选自下组的氨基酸残基处包含氨基酸取代：(1)I61、V63、E77、E84、V93、L96、K98、N100和V132；(2)I61、E77、Q82、K83和E84；(3)I61、V63、K83、E84和V132；(4)I61、E77、E84、R107和V132；(5)I61、V63、E77、K83、E84和N100；(6)I61、E77、Q82、K83、E84和R107；(7)I61、E77、Q82、E84、V93、L96、N100、R107、G109和V132；(8)I61、E77、Q82、K83、E84和V132；(9)I61；(10)I61、D95、L96、G109和V132；(11)I61、D95、L96、K98、G109和V132；(12)I61、E77、E84、V93、R107和V132；(13)E77、L96、N100、G109和V132；(14)I61、V63、Q82、E84、D95、L96、N100和V132；(15)I61、E77、Q82、K83、E84、V93、D95、L96、K98、N100和V132；(16)I61、E77、Q82、K83、E84和V93；(17)I61、V63、E77、K83、E84、D95、L96、K98和N100；(18)I61、V63、E77、K83、D95、L96、K98、N100和G109；(19)I61、E77、Q82、E84、V93、D95、L96、K98和N100；和，(20)I61、V63、E77、Q82和E84。
在本申请中，所述突变体可以包含选自下组的一个或多个(例如，1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个或更多个)氨基酸取代：R22C、I29L、I61L/V/F、V63I、E77I/N/Q/K/H/M/R/N/V/L、Q82S/R/G/N、K83R、E84K/H/D/R/G、V93L/A、D95H/R/E、D96S/T、K98R、N100G/K/D/E、R107N/S、G109R/H和V132L/R/I/S。
在本申请中，氨基酸取代“XnY/Z”是指相应于SEQ ID NO:50所示氨基酸序列中第n位的残基X被取代为氨基酸残基Y或者氨基酸残基Z，其中n为正整数，X、Y和Z分别独立地为任意氨基酸残基的缩写，且X不同于Y或Z。例如，氨基酸取代“I61L/V/F”是指相应于SEQ ID NO:50所示氨基酸序列中第61位的残基I被取代为氨基酸残基L、V或F。
在本申请中，所述突变体可以包含选自下组的氨基酸取代：(1)I61L、V63I、E77I、E84K、V93L、L96S、K98R、N100G和V132L；(2)I61V、E77N、Q82S、K83R和E84H；(3)I61F、V63I、K83R、E84K和V132I；(4)I61L、E77Q、E84D、R107N和V132I；(5)I61L、V63I、E77K、K83R、E84D和N100G；(6)I61V、E77H、Q82R、K83R、E84H和R107S；(7)I61L、E77I、Q82G、E84R、V93L、L96T、N100G、R107S、G109R和V132R；(8)I61L、E77M、Q82G、K83R、E84D和V132L；(9)I61L；(10)I61F、D95H、L96S、G109H和V132S；(11)I61F、D95H、L96S、K98R、G109H和V132S；(12)I61L、E77Q、E84D、V93A、R107N和V132I；(13)E77K、L96S、N100K、G109H和V132L；(14)I61L、V63I、Q82G、E84G、D95R、L96S、N100D和V132I；(15)I61L、E77R、Q82N、K83R、E84G、V93L、D95E、L96T、K98R、N100D和V132L；(16)I61V、E77N、Q82S、K83R、E84H和V93A；(17)I61V、V63I、E77V、K83R、E84D、D95E、L96T、K98R和N100E；(18)I61L、V63I、E77V、K83R、D95E、L96S、K98R、N100D和G109R；(19)I61V、E77L、Q82G、E84G、V93L、D95E、L96T、K98R和N100G；和，(20)I61L、V63I、E77N、Q82G和E84G。
在本申请中，在人SIRPα变体1(如SEQ ID NO:50所示的氨基酸序列，即人SIRPα的氨基酸序列中的第33-149位残基)的基础上，分别包含以上(1)-(20)的氨基酸取代组的SIRPα变体1的突变体可依次被命名为M1、M5、M12、M35、M37、M41、M57、M67、M81、M82、M84、M91、M99、M102、M111、M122、M126、M130、M135和M145。所述SIRPα变体1的突变体可依次包含如SEQ ID NO:51-SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述SIRPα变体1的突变体是M91,所述SIRPα变体1的突变体包含如SEQ ID NO:62所示的氨基酸序列。
特异性结合肿瘤相关抗原的第一结合域
在本申请中，所述第一结合域可以包含抗体或其抗原结合片段或变体。例如，所述抗体可以选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗原结合片段选自下组：Fab，Fab'，(Fab')2，F(ab)2，dAb，分离的互补决定区CDR，Fv和scFv。
在本申请中，所述抗体或其抗原结合片段的所述变体可以在所述抗体或所述其抗原结合片段中经过取代、缺失或添加一个或多个(例如，1-2个、1-3个、1-4个、1-5个、1-6个、1-7个、1-8个、1-9个、1-10个或更多个)氨基酸的蛋白质或多肽。或者所述抗体或其抗原结合片段的所述变体可以与所述抗体或所述其抗原结合片段具有至少90％(例如，例如，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或至少100％)序列同源性的蛋白质或多肽。
特异性结合CD38的第一结合域
本申请所述的抗体或其抗原结合片段或变体，可通过与CD38蛋白特异性结合而杀伤肿瘤细胞和/或抑制肿瘤生长。例如，所述肿瘤可以包括CD38阳性的肿瘤。如，所述CD38阳性的肿瘤可以选自以下组：骨髓瘤、淋巴瘤和白血病。又例如，所述肿瘤选自以下组：非霍奇金淋巴瘤和霍奇金淋巴瘤。所述肿瘤的细胞可以选自下组：Raji细胞、Daudi细胞、Ramos细胞和RPMI8226细胞。在本申请中，所述抗体、其抗原结合片段或变体可以杀伤多发性骨髓瘤、淋巴瘤、白血病、非霍奇金淋巴瘤和霍奇金淋巴瘤细胞或抑制多发性骨髓瘤、淋巴瘤、白血病、非霍奇金淋巴瘤和霍奇金淋巴瘤生长。
本申请所述的CD38蛋白可为人CD38蛋白或其功能片段。例如，所述的CD38蛋白可以不为小鼠CD38蛋白，或者可以不为大鼠CD38蛋白。在某些实施方式中，本申请所述的抗体、其抗原结合片段或变体基本上不结合小鼠CD38蛋白或大鼠CD38蛋白。
本申请所述的抗体、其抗原结合片段或变体，可以与参比抗体竞争结合所述CD38蛋白。所述参比抗体可以包含轻链可变区和重链可变区。例如，所述参比抗体的轻链可变区可包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3。所述参比抗体的重链可变区可包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6。
例如，所述参比抗体的轻链可变区的氨基酸序列可以为：SEQ ID NO:7，且所述参比抗体的重链可变区的氨基酸序列可以为：SEQ ID NO:8。又例如，所述参比抗体的轻链可包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:18和SEQ ID NO:20；且所述参比抗体的重链可包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:19和SEQ ID NO:21。例如，所述参比抗体的轻链可包含如SEQ ID NO:11所示的氨基酸序列，且所述参比抗体的重链可包含如SEQ ID NO:13所示的氨基酸序列。例如，所述参比抗体的轻链可包含如SEQ ID NO:16所示的氨基酸序列，且所述参比抗体的重链可包含如SEQ ID NO:17所示的氨基酸序列。例如，所述参比抗体的轻链可包含如SEQ ID NO:18所示的氨基酸序列，且所述参比抗体的重链可包含如SEQ ID NO:19所示 的氨基酸序列。例如，所述参比抗体的轻链可包含如SEQ ID NO:20所示的氨基酸序列，且所述参比抗体的重链可包含如SEQ ID NO:21所示的氨基酸序列。
本申请所述的抗体、其抗原结合片段或变体可包含抗体轻链或其片段。例如，所述抗体轻链或其片段可包括Igκ恒定区，例如可包含人Igκ恒定区。
例如，所述抗体轻链或其片段可包含LCDR1，且所述LCDR1可包含如下的氨基酸序列：SEQ ID NO:1。所述抗体轻链或其片段可包含LCDR2，且所述LCDR2可包含如下的氨基酸序列：SEQ ID NO:2。所述抗体轻链或其片段可包含LCDR3，且所述LCDR3可包含如下的氨基酸序列：SEQ ID NO:3。
本申请所述抗体的轻链或其片段可包含轻链可变区VL，且所述轻链可变区VL的氨基酸序列可以为：SEQ ID NO:7。在某些实施方式中，所述抗体轻链或其片段可包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:18和SEQ ID NO:20。
本申请所述的抗体或其抗原结合片段可包含抗体重链或其片段。例如，所述抗体重链或其片段还包含人恒定区。其中，所述人恒定区可包括人IgG恒定区。其中，所述IgG恒定区可包含人IgG1恒定区或IgG4。
例如，所述抗体重链或其片段可包含HCDR1，且所述HCDR1可包含如下的氨基酸序列：SEQ ID NO:4。所述抗体重链或其片段可包含HCDR2，且所述HCDR2可包含如下的氨基酸序列：SEQ ID NO:5。又例如，所述抗体重链或其片段可包含HCDR3，且所述HCDR3可包含如下的氨基酸序列：SEQ ID NO:6。
所述抗体重链或其片段可包含重链可变区VH，且所述重链可变区VH可包含如下的氨基酸序列：SEQ ID NO:8。在某些实施方式中，所述抗体重链可包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:19和SEQ ID NO:21。
在某些实施方式中，本申请所述的抗体或其抗原结合片段中LCDR1的氨基酸序列可包括SEQ ID NO:1；LCDR2的氨基酸序列可包括SEQ ID NO:2；LCDR3的氨基酸序列可包括SEQ ID NO:3；且HCDR1的氨基酸序列可包括SEQ ID NO:4或；HCDR2的氨基酸序列可包括SEQ ID NO:5；HCDR3的氨基酸序列可包括SEQ ID NO:6。例如，该抗体或其抗原结合片段可包括抗体SG003或与其具有相同的LCDR1-3及HCDR1-3的抗体。在某些实施方式中，本申请所述的抗体或其抗原结合片段的轻链可包含轻链可变区，所述轻链可变区的氨基酸序列可包括SEQ ID NO:7；且其中重链可包含重链可变区，所述重链可变区的氨基酸序列可包括SEQ ID NO:8。例如，该抗体或其抗原结合片段可包括抗体SG003或与其具有相同的轻链可变区及重链可变区的抗体。在某些实施方式中，本申请所述的抗体或其抗原结合片段可包含轻链和重链，所述轻链的轻链氨基酸序列如SEQ ID NO:11所示且所述重链氨基酸序列如 SEQ ID NO:13所示。例如，该抗体或其抗原结合片段可包括抗体SG003或与其具有相同的轻链及重链氨基酸序列。
在某些实施方式中，本申请所述的抗体可以为SG003。抗体SG003的LCDR1-3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示；VL的氨基酸序列如SEQ ID NO:7所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；VH的氨基酸序列如SEQ ID NO:8所示；轻链的氨基酸序列如SEQ ID NO:11所示；重链的氨基酸序列如SEQ ID NO:13所示。
本申请所述的抗体或其抗原结合片段或变体，还可以在SG003的轻链和/或重链的氨基酸序列中包含一个或多个随机突变(例如，一个或多个、如一个或数个氨基酸取代)。例如，所述抗体、其抗原结合片段或变体可在SG003的轻链可变区的框架区L-FR1-4的一个或多个位点包含一个或多个随机突变(例如，一个或多个，如一个或数个氨基酸取代)，和/或在SG003的重链可变区的框架区H-FR1-4的一个或多个位点包含一个或多个随机突变(例如，一个或多个，如一个或数个氨基酸取代)。例如，发生随机突变后，所述的抗体或其抗原结合片段或变体的轻链可包含包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:16、SEQ ID NO:18和SEQ ID NO:20；和/或所述的抗体或其抗原结合片段或变体的重链可包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:17、SEQ ID NO:19和SEQ ID NO:21。发生随机突变后的CD38抗体或其抗原结合片段或变体，仍具有特异性结合人CD38蛋白的能力。
在某些实施方式中，本申请所述的抗体或其抗原结合片段的轻链的氨基酸序列包括SEQ ID NO:11；并且其重链的氨基酸序列包括SEQ ID NO:13；或者本申请所述的抗体或其抗原结合片段的轻链的氨基酸序列包括SEQ ID NO:16；并且其重链的氨基酸序列包括SEQ ID NO:17；或者，本申请所述的抗体或其抗原结合片段的轻链的氨基酸序列包括SEQ ID NO:18；并且其重链的氨基酸序列包括SEQ ID NO:19；或者，本申请所述的抗体或其抗原结合片段的轻链的氨基酸序列包括SEQ ID NO:20；并且其重链的氨基酸序列包括SEQ ID NO:21。
特异性结合AXL的第一结合域
本申请所述的抗体或其抗原结合片段或变体，可通过与AXL特异性结合而杀伤肿瘤细胞和/或抑制肿瘤生长。例如，所述AXL可以为人AXL。
本申请所述的抗体、其抗原结合片段或变体，可以与参比抗体竞争结合所述AXL蛋白。所述参比抗体可以包含轻链可变区和重链可变区。例如，所述参比抗体的轻链可变区可包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24。所述参比抗体的重链可变区可包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27。
例如，所述参比抗体的轻链可变区的氨基酸序列可以为：SEQ ID NO:28，且所述参比抗体的重链可变区的氨基酸序列可以为：SEQ ID NO:29。又例如，所述参比抗体的轻链的氨基酸序列可以为：SEQ ID NO:32；且所述参比抗体的重链的氨基酸序列可以为：SEQ ID NO:34。
本申请所述的抗体、其抗原结合片段或变体可包含抗体轻链或其片段。例如，所述抗体轻链或其片段可包括Igκ恒定区，例如可包含人Igκ恒定区。
例如，所述抗体轻链或其片段可包含LCDR1，且所述LCDR1可包含如下的氨基酸序列：SEQ ID NO:22。所述抗体轻链或其片段可包含LCDR2，且所述LCDR2可包含如下的氨基酸序列：SEQ ID NO:23。所述抗体轻链或其片段可包含LCDR3，且所述LCDR3可包含如下的氨基酸序列：SEQ ID NO:24。
本申请所述抗体的轻链或其片段可包含轻链可变区VL，且所述轻链可变区VL的氨基酸序列可以为：SEQ ID NO:28。在某些实施方式中，所述抗体轻链或其片段的氨基酸序列可以为：SEQ ID NO:32。
本申请所述的抗体或其抗原结合片段可包含抗体重链或其片段。例如，所述抗体重链或其片段还包含人恒定区。其中，所述人恒定区可包括人IgG恒定区。其中，所述IgG恒定区可包含人IgG1恒定区或IgG4。
例如，所述抗体重链或其片段可包含HCDR1，且所述HCDR1可包含如下的氨基酸序列：SEQ ID NO:25。所述抗体重链或其片段可包含HCDR2，且所述HCDR2可包含如下的氨基酸序列：SEQ ID NO:26。又例如，所述抗体重链或其片段可包含HCDR3，且所述HCDR3可包含如下的氨基酸序列：SEQ ID NO:27。
所述抗体重链或其片段可包含重链可变区VH，且所述重链可变区VH的氨基酸序列可以为：SEQ ID NO:29。在某些实施方式中，所述抗体重链或其片段的氨基酸序列可以为：SEQ ID NO:34。
在某些实施方式中，本申请所述的抗体或其抗原结合片段中LCDR1的氨基酸序列可包括SEQ ID NO:22或其变体；LCDR2的氨基酸序列可包括SEQ ID NO:23或其变体；LCDR3的氨基酸序列可包括SEQ ID NO:24或其变体；且HCDR1的氨基酸序列可包括SEQ ID NO:25或其变体；HCDR2的氨基酸序列可包括SEQ ID NO:26或其变体；HCDR3的氨基酸序列可包括SEQ ID NO:27或其变体。例如，该抗体或其抗原结合片段可包括抗体C6G12或与其具有相同的LCDR1-3及HCDR1-3的抗体。在某些实施方式中，本申请所述的抗体或其抗原结合片段的轻链可包含轻链可变区，所述轻链可变区的氨基酸序列可包括SEQ ID NO:28或其变体；且其中重链可包含重链可变区，所述重链可变区的氨基酸序列可包括SEQ ID NO:29或其变体。例如，该抗体或其抗原结合片段可包括抗体C6G12或与其具有相同的轻链可变区及 重链可变区的抗体。在某些实施方式中，本申请所述的抗体或其抗原结合片段可包含轻链和重链，所述轻链的轻链氨基酸序列如SEQ ID NO:32所示且所述重链氨基酸序列如SEQ ID NO:34所示。例如，该抗体或其抗原结合片段可包括抗体C6G12或与其具有相同的轻链及重链氨基酸序列。
在某些实施方式中，本申请所述的抗体可以为C6G12。抗体C6G12的LCDR1-3的氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示；VL的氨基酸序列如SEQ ID NO:28所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示；VH的氨基酸序列如SEQ ID NO:29所示；轻链的氨基酸序列如SEQ ID NO:32所示；重链的氨基酸序列如SEQ ID NO:34所示。
特异性结合Trop2的第一结合域
本申请所述的抗体或其抗原结合片段或变体，可通过与Trop2特异性结合而杀伤肿瘤细胞和/或抑制肿瘤生长。例如，所述Trop2可以为人Trop2。
本申请所述的抗体、其抗原结合片段或变体，可以与参比抗体竞争结合所述Trop2蛋白。所述参比抗体可以包含轻链可变区和重链可变区。例如，所述参比抗体的轻链可变区可包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38。所述参比抗体的重链可变区可包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41。
例如，所述参比抗体的轻链可变区的氨基酸序列可以为：SEQ ID NO:42，且所述参比抗体的重链可变区的氨基酸序列可以为：SEQ ID NO:43。又例如，所述参比抗体的轻链可包的氨基酸序列可以为：SEQ ID NO:46；且所述参比抗体的重链的氨基酸序列可以为：SEQ ID NO:48。例如，所述参比抗体的轻链可包含如SEQ ID NO:46所示的氨基酸序列，且所述参比抗体的重链可包含如SEQ ID NO:48所示的氨基酸序列。
本申请所述的抗体、其抗原结合片段或变体可包含抗体轻链或其片段。例如，所述抗体轻链或其片段可包括Igκ恒定区，例如可包含人Igκ恒定区。
例如，所述抗体轻链或其片段可包含LCDR1，且所述LCDR1可包含如下的氨基酸序列：SEQ ID NO:36。所述抗体轻链或其片段可包含LCDR2，且所述LCDR2可包含如下的氨基酸序列：SEQ ID NO:37。所述抗体轻链或其片段可包含LCDR3，且所述LCDR3可包含如下的氨基酸序列：SEQ ID NO:38。
本申请所述抗体的轻链或其片段可包含轻链可变区VL，且所述轻链可变区VL可包含如下的氨基酸序列：SEQ ID NO:42。在某些实施方式中，所述抗体轻链或其片段的氨基酸序列可以为：SEQ ID NO:46。
本申请所述的抗体或其抗原结合片段可包含抗体重链或其片段。例如，所述抗体重链或其片段还包含人恒定区。其中，所述人恒定区可包括人IgG恒定区。其中，所述IgG恒定区可包含人IgG1恒定区或IgG4。
例如，所述抗体重链或其片段可包含HCDR1，且所述HCDR1可包含如下的氨基酸序列：SEQ ID NO:39。所述抗体重链或其片段可包含HCDR2，且所述HCDR2可包含如下的氨基酸序列：SEQ ID NO:40。又例如，所述抗体重链或其片段可包含HCDR3，且所述HCDR3可包含如下的氨基酸序列：SEQ ID NO:41。
所述抗体重链或其片段可包含重链可变区VH，且所述重链可变区VH可包含如下的氨基酸序列：SEQ ID NO:43。在某些实施方式中，所述抗体重链或其片段的氨基酸序列可以为：SEQ ID NO:48。
在某些实施方式中，本申请所述的抗体或其抗原结合片段中LCDR1的氨基酸序列可包括SEQ ID NO:36或其变体；LCDR2的氨基酸序列可包括SEQ ID NO:37或其变体；LCDR3的氨基酸序列可包括SEQ ID NO:38或其变体；且HCDR1的氨基酸序列可包括SEQ ID NO:39或其变体；HCDR2的氨基酸序列可包括SEQ ID NO:40或其变体；HCDR3的氨基酸序列可包括SEQ ID NO:41或其变体。例如，该抗体或其抗原结合片段可包括抗体SG701或与其具有相同的LCDR1-3及HCDR1-3的抗体。在某些实施方式中，本申请所述的抗体或其抗原结合片段的轻链可包含轻链可变区，所述轻链可变区的氨基酸序列可包括SEQ ID NO:42或其变体；且其中重链可包含重链可变区，所述重链可变区的氨基酸序列可包括SEQ ID NO:43或其变体。例如，该抗体或其抗原结合片段可包括抗体SG701或与其具有相同的轻链可变区及重链可变区的抗体。在某些实施方式中，本申请所述的抗体或其抗原结合片段可包含轻链和重链，所述轻链的轻链氨基酸序列如SEQ ID NO:46所示且所述重链氨基酸序列如SEQ ID NO:48所示。例如，该抗体或其抗原结合片段可包括抗体SG701或与其具有相同的轻链及重链氨基酸序列。
在某些实施方式中，本申请所述的抗体可以为SG701。抗体SG701的LCDR1-3的氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38所示；VL的氨基酸序列如SEQ ID NO:42所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示；VH的氨基酸序列如SEQ ID NO:43所示；轻链的氨基酸序列如SEQ ID NO:46所示；重链的氨基酸序列如SEQ ID NO:48所示。
第一结合域和第二结合域的连接
在本申请中，所述第一结合域可以位于所述第二结合域的N端。例如，所述第一结合域 的C端可以通过连接子间接连接于所述第二结合域的N端。在某些情形中，所述第一结合域的C端也可以直接(例如，在框内)连接于第二结合域的N端。
在本申请中，所述融合蛋白还可以包含连接子，所述连接子可以位于所述第一结合域的C端且位于所述第二结合域的N端。例如，在所述融合蛋白中，第一结合域的C端可以连接连接子的N端，连接子的C端可以连接第二结合域的N端。例如，在所述融合蛋白中，从N端到C端，可以依次包含第一结合域、连接子和第二结合域。
在本申请中，所述连接子可以包含如SEQ ID NO:73-74中任一项所示的氨基酸序列。
在某些情形中，所述融合蛋白可以包含至少2个(例如，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，至少8个，至少9个，至少10个，或更多)所述第二结合域。在本申请中，所述每个所述第二结合域可以分别位于所述第一结合域的C端。在本申请中，所述2个以上的第二结合域可以分别直接或间接连接于所述第一结合域的C端。
在本申请中，所述融合蛋白可以包含特异性结合CD38抗原的第一结合域，以及特异性结合CD47蛋白的第二结合域，其中所述第一结合域可以包含特异性结合CD38抗原的抗体或其抗原结合片段或变体，第二结合域可以包含人SIRPα变体1的突变体，特异性结合CD38抗原的抗体或其抗原结合片段或变体的C端可以直接或者间接连接人SIRPα变体1的突变体的N端。例如，所述第二结合域可以包含至少2个人SIRPα变体1的突变体，且2个人SIRPα变体1的突变体的N端分别连接特异性结合CD38抗原的抗体或其抗原结合片段或变体的C端。
例如，如图1所示，所述融合蛋白(SG3847L1)的第一结合域可以包含SG003，第二结合域可以包含2个SIRPα变体1的突变体M91，采用的连接子1的序列如SEQ ID NO:73所示，2个M91的N端分别通过连接子1连接于SG003的2条重链的C端，在所述融合蛋白中，M91连接SG003重链的C端可以得到第二多肽链，SG003的轻链可以命名为第一多肽链。SG3847L1的所述第二多肽链和所述第一多肽链的氨基酸序列分别如SEQ ID NO:75和SEQ ID NO:11所示。
例如，如图1所示，所述融合蛋白(SG3847L2)的第一结合域可以包含SG003，第二结合域可以包含2个SIRPα变体1的突变体M91，采用的连接子2的序列如SEQ ID NO:74所示，2个M91的N端分别通过连接子2连接于SG003的2条重链的C端，在所述融合蛋白中，M91连接SG003重链的C端可以得到第二多肽链，SG003的轻链可以命名为第一多肽链。SG3847L2的所述第二多肽链和所述第一多肽链的氨基酸序列分别如SEQ ID NO:76和SEQ ID NO:11所示。
在本申请中，所述融合蛋白可以包含特异性结合Trop2抗原的第一结合域，以及特异性 结合CD47蛋白的第二结合域，其中所述第一结合域可以包含特异性结合Trop2抗原的抗体或其抗原结合片段或变体，第二结合域可以包含人SIRPα变体1的突变体，特异性结合Trop2抗原的抗体或其抗原结合片段或变体的C端可以直接或者间接连接人SIRPα变体1的突变体的N端。在本申请中，所述第二结合域可以包含至少2个人SIRPα变体1的突变体，且2个人SIRPα变体1的突变体的N端分别连接特异性结合AXL抗原的抗体或其抗原结合片段或变体的C端。
例如，如图1所示，所述融合蛋白(SGTrop247)的第一结合域可以包含抗体SG701，第二结合域可以包含2个SIRPα变体1的突变体M91，采用的连接子的序列如SEQ ID NO:74所示，2个M91的N端分别通过连接子连接于SG701的2条重链的C端，在所述融合蛋白中，M91连接SG701重链的C端可以得到第二多肽链，SG701的轻链可以命名为第一多肽链。SGTrop247的所述第二多肽链和所述第一多肽链的氨基酸序列分别如SEQ ID NO:78和SEQ ID NO:46所示。
在本申请中，所述融合蛋白可以包含特异性结合AXL抗原的第一结合域，以及特异性结合CD47蛋白的第二结合域，其中所述第一结合域可以包含特异性结合AXL抗原的抗体或其抗原结合片段或变体，第二结合域可以包含人SIRPα变体1的突变体，特异性结合AXL抗原的抗体或其抗原结合片段或变体的C端可以直接或者间接连接人SIRPα变体1的突变体的N端。在本申请中，所述第二结合域可以包含至少2个人SIRPα变体1的突变体，且2个人SIRPα变体1的突变体的N端分别连接特异性结合AXL抗原的抗体或其抗原结合片段或变体的C端。
例如，如图1所示，所述融合蛋白(SGAXL47)的第一结合域可以包含抗体C6G12，第二结合域可以包含2个SIRPα变体1的突变体M91，采用的连接子的序列如SEQ ID NO:74所示，2个M91的N端分别通过连接子连接于C6G12的2条重链的C端，在所述融合蛋白中，M91连接C6G12重链的C端可以得到第二多肽链，C6G12的轻链可以命名为第一多肽链。SGAXL47的所述第二多肽链和所述第一多肽链的氨基酸序列分别如SEQ ID NO:77和SEQ ID NO:32所示。
核酸分子、载体和细胞及制备方法
另一方面，本申请提供一个或多个分离的核酸分子，其编码所述的融合蛋白或者所述的免疫缀合物。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗体或其抗原结合片段，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
重组DNA和分子克隆技术包括由Sambrook,J.,Fritsch,E.F.和Maniatis,T.Molecular Cloning:A Laboratory Manual；Cold Spring Harbor Laboratory Press:Cold Spring Harbor,(1989)(Maniatis)和由T.J.Silhavy,M.L.Bennan和L.W.Enquist,Experiments with Gene Fusions,Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.(1984)以及由Ausubel,F.M.等，Current Protocols in Molecular Biology,pub.by Greene Publishing Assoc.and Wiley-Interscience(1987)描述的那些技术。简而言之，可从基因组DNA片段、cDNA和RNA制备所述核酸，所有这些核酸可直接从细胞中提取或通过各种扩增方法(包括但不限于PCR和RT-PCR)重组产生。
核酸的直接化学合成通常涉及将3'-封闭的和5'-封闭的核苷酸单体依次添加至生长中的核苷酸聚合物链的末端5'-羟基，其中每次添加通过亲核攻击所添加的单体的3'-位上的生长链的末端5'-羟基来实现，所述单体通常是磷衍生物，诸如磷酸三酯、亚磷酰胺等。参见，例如，Matteuci等，Tet.Lett.521:719(1980)；属于Caruthers等的美国专利第4,500,707号；和属于Southern等的美国专利第5,436,327号和第5,700,637号；在另一方面，本申请提供了包含本申请的分离的多核苷酸的载体。所述载体可以是任何线性核酸、质粒、噬菌粒、粘粒、RNA载体、病毒载体等。病毒载体的非限制性实例可包括逆转录病毒、腺病毒和腺相关病毒。在一些实施方案中，所述载体是表达载体，例如，噬菌体展示载体。
另一方面，本申请提供一个或多个载体，其包含所述的核酸分子。例如，所述载体可以包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA 启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。在某些实施方式中，所述载体可以为表达载体。
所述载体还可含有一个或多个选择标记基因，其在表达后赋予可用于选择或以其它方式鉴定携带载体的宿主细胞的一个或多个表型性状。用于真核细胞的合适的选择标记的非限制性实例包括二氢叶酸还原酶和新霉素抗性。
另一方面，本申请提供一种细胞，所述细胞包含所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，或所述的载体。所述细胞可以是宿主细胞。例如，所述细胞可以包括如下许多细胞类型，如大肠杆菌或枯草菌等原核细胞，如酵母细胞或曲霉菌等真菌细胞，如S2果蝇细胞或Sf9等昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞的动物细胞。
例如，可通过多种已建立的技术将所述载体稳定地或瞬时引入宿主细胞。例如，一种方法涉及氯化钙处理，其中通过钙沉淀引入载体。也可以按照类似方法使用其它盐，例如磷酸钙。另外，可以用电穿孔(即，施加电流以增加细胞对核酸的渗透性)。转化方法的其它实例包括显微注射、DEAE葡聚糖介导的转化和在乙酸锂存在下的热休克。脂质复合物、脂质体和树状聚合物也可用于转染宿主细胞。
在将异源序列引入宿主细胞中时，可实施多种方法来鉴定已向其中引入了载体的宿主细胞。一种示例性选择方法包括将单个细胞继代培养以形成单个菌落，然后测试所需蛋白质产物的表达。另一种方法需要基于通过载体内包含的选择标记基因的表达赋予的表型性状选择含有异源序列的宿主细胞。
例如，将本申请的各种异源序列引入宿主细胞可通过方法诸如PCR、Southern印迹或Northern印迹杂交来确认。例如，可从所得宿主细胞制备核酸，并且可使用对目标序列是特异的引物，通过PCR扩增特定目标序列。将扩增产物进行琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳或毛细管电泳，然后用溴化乙锭、SYBR Green溶液等染色，或用UV检测来检测DNA。或者，可在杂交反应中使用对目标序列是特异的核酸探针。特定基因序列的表达可以通过与PCR，Northern印迹杂交或通过使用与编码的基因产物反应的抗体的免疫测定的反转录检测相应的mRNA来确定。示例性免疫测定包括但不限于ELISA、放射免疫测定和夹心免疫测定。
此外，将本申请的各种异源序列引入宿主细胞可以通过异源序列编码的酶(例如，酶促 标志物)的酶促活性来确认实。可通过本领域已知的多种方法测定酶。通常，酶促活性可通过产物的形成或正在研究的酶促反应的底物的转化来确定。所述反应可在体外或体内进行。
另一方面，本申请提供一种制备所述的融合蛋白的方法，其可以包括在使得所述融合蛋白能够表达的条件下培养所述的细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
在某些情形中，所述方法还可包括分离和/或纯化所述融合蛋白的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的融合蛋白。
免疫缀合物、组合物和应用
另一方面，本申请提供一种免疫缀合物，其包含所述的融合蛋白。例如，所述免疫缀合物可以为融合蛋白-药物缀合物(ADC)，其中本申请所述的融合蛋白与一种或多种治疗剂缀合，所述治疗剂包括但不限于细胞毒性剂、放射性毒性剂(例如，放射性同位素)和/或免疫抑制剂(例如，通过抑制免疫应答等途径杀伤细胞的任何药剂)等。在某些实施方式中，所述治疗剂可以为能够治疗肿瘤相关的疾病或病症的治疗剂。
所述缀合可通过肽连接子(例如，可切割连接子)或其它方式进行，例如，所述连接子可以为酸不稳定连接子、肽酶敏感连接子、光不稳定连接子等。
另一方面，本申请提供一种组合物，所述组合物包含所述的融合蛋白，所述的免疫缀合物，或所述的核酸分子，以及任选地药学上可接受的赋形剂。
例如，所述药学上可接受的赋形剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
在本申请中，可按照本领域的常规技术手段将所述组合物与药学上可接受的载体或稀释剂以及任何其他已知的辅剂和赋形剂配制在一起，例如按照Remington：The Science and Practice of Pharmacy，第十九版，Gennaro编辑，Mack Publishing Co.，Easton，PA，1995中公开的技术进行操作。
在本申请中，所述组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
例如，所述组合物可以用于抑制肿瘤生长。例如，本申请的组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
例如，本申请所述的组合物可以为适于口服给药的形式，如片剂，胶囊剂，丸剂，粉剂，缓释制剂，溶液剂，混悬剂，或者用于肠胃外注射，如无菌溶液剂，混悬剂或乳剂，或者用 于软膏或乳膏局部给药或作为栓剂直肠给药。所述组合物可以是适合精确剂量单次给药的单位剂量形式。所述组合物可以进一步包含常规的药物载体或赋形剂。此外，所述组合物可以包括其他药物或药剂，载体，佐剂等。
本申请所述的组合物可以包含治疗有效量的所述融合蛋白。所述治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的病症或病症(例如肿瘤)和/或其任何并发症而所需的剂量。所述剂量的具体量/浓度可以根据施用方法和患者需要而变化，并且可以基于例如患者体积，粘度和/或体重等来确定。例如，合适的剂量可以是约0.1mg或1mg/kg/天至约50mg/kg/天；有时，剂量可能会更高。应当理解的是，基于特定患者，制剂和/或疾病的状况，本领域技术人员(例如，医生或药剂师)可以方便地调整这些特定剂量。
在本申请中，术语“治疗”或“医治”或“缓解”或“改善”在本申请中可互换使用，并且是指获得有益或所需的结果(包括但不限于治疗益处和/或预防益处)的方法。在本申请中，治疗益处通常是指根除或减轻所治疗的潜在病症的严重性。此外，通过根除、减轻严重性或减少与潜在病症相关的一种或多种生理症状的发生率，以使得在受试者中观察到改善(尽管受试者仍然可能受到潜在病症折磨)来实现治疗益处。对于预防益处，可向处于发展特定疾病的风险中的受试者，或报告疾病的一种或多种生理症状的受试者施用组合物，即使可能尚未进行该疾病的诊断。
另一方面，本申请提供了所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞在制备药物中的用途，其中所述药物可以用于治疗肿瘤或自免疫疾病。
另一方面，本申请所述的融合蛋白、免疫缀合物、核酸分子、载体、组合物或细胞可以用于治疗所述肿瘤或自免疫疾病。
另一方面，本申请提供了治疗肿瘤或自免疫疾病的方法，所述方法包括向受试者施用本申请所述的融合蛋白、免疫缀合物、核酸分子、载体、组合物或细胞。
另一方面，本申请提供了一种阻断CD47蛋白与SIRPα相互作用的方法，所述方法可包括施用(例如，向有需要的受试者或细胞或生物学样品施用)本申请所述的融合蛋白或组合物。
另一方面，本申请提供了一种抑制肿瘤或肿瘤细胞生长和/或增殖的方法，所述方法可包括使本申请所述的融合蛋白或组合物与所述肿瘤或肿瘤细胞接触。例如，所述接触可在体外发生。
在某些实施方式中，所述肿瘤可以包括非实体瘤和实体瘤。
在某些实施方式中，所述肿瘤可以包括多发性骨髓瘤、白血病、非霍奇金淋巴瘤、霍奇 金淋巴瘤、神经胶质瘤、生殖细胞瘤、肉瘤、见皮瘤、胎盘瘤、脑癌、骨癌、皮肤癌、鼻咽癌、肺癌、口腔癌、食道癌、胃癌、肝癌、胰腺癌、前列腺癌、肠癌、乳腺癌、宫颈癌、卵巢癌和睾丸癌。
在某些实施方式中，所述自免疫疾病可以包括慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征(goodpasture syndrome)、寻常天皰疮、类天皰疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症、急性特发性多神经炎、系统性红斑狼疮、类风湿性关节炎、硬皮病和结节性多动脉炎。
另一方面，本申请提供了所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞，其可以治疗肿瘤或自免疫疾病。
另一方面，本申请提供了一种可以阻断CD47蛋白与SIRPα相互作用的方法，其可以包括向有需要的受试者施用有效量的所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞。
在本申请中，术语“受试者”通常是指人或非人动物，包括但不限于猫、狗、马、猪、牛、绵羊、山羊、兔、小鼠、大鼠或猴。
另一方面，本申请提供了一种可以抑制肿瘤或肿瘤细胞生长和/或增殖的方法，其可以包括向有需要的受试者施用有效量的所述的融合蛋白，所述的免疫缀合物，所述的核酸分子，所述的载体，所述的组合物，或所述的细胞。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1.不同连接子选择对活性的影响
1.1采用不同连接子制备融合蛋白
参照如图1所示的融合蛋白结构，以抗CD38人源化抗体SG003、SIRPα变体1的突变体M91(SEQ ID:NO 62)为例，从N端到C端，选用不同的连接子，依次连接SG003、连接子和2个M91，其中2个M91的N端分别和SG003的重链的C端连接，研究不同的连接子对融合蛋白生物学活性的影响。
抗体SG003的LCDR1-3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示；VL的氨基酸序列如SEQ ID NO:7所示；编码VL的核苷酸序列如SEQ ID NO:9 所示；抗体SG003的HCDR1-3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；VH的氨基酸序列如SEQ ID NO:8所示；编码VH的核苷酸序列如SEQ ID NO:10所示。抗体SG003的轻链的氨基酸序列如SEQ ID NO:11所示；编码其的核苷酸序列如SEQ ID NO:12所示。抗体SG003的重链的氨基酸序列如SEQ ID NO:13所示；编码其的核苷酸序列如SEQ ID NO:14所示。
选择连接子1(SEQ ID:NO 73)，构建的融合蛋白命名为SG3847L1，所述SG3847L1由第一多肽链和第二多肽链构成，第一多肽链即SG003的轻链，其氨基酸序列如SEQ ID NO:11所示，第二多肽链即SG003的重链和M91通过连接子1连接得到的多肽链，其氨基酸序列如SEQ ID NO:75所示。
选择连接子2(SEQ ID:NO 74)，构建的融合蛋白命名为SG3847L2，所述SG3847L2由第一多肽链和第二多肽链构成，第一多肽链即SG003的轻链，其氨基酸序列如SEQ ID NO:11所示，第二多肽链即SG003的重链和M91通过连接子2连接得到的多肽链，其氨基酸序列如SEQ ID NO:76所示。
1.2分别与双抗原的结合活性分析
(1)以SG003为对照，ELISA评价融合蛋白和CD38结合活性。
将靶抗原CD38-His(1μg/ml)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的SG003抗体、SG3847L1或SG3847L2融合蛋白，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG二抗(Goat Anti human IgG HRP，Thermo Fisher Scientific)，37℃反应30分钟；PBST洗涤5次；每孔加入100μl TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析融合蛋白与靶抗原CD38结合能力。
如图2所示，SG3847L1、SG3847L2和SG003抗体相比，与靶抗原CD38的结合力相似，SG3847L1、SG3847L2和SG003的EC50值分别为0.175nM、0.149nM和0.133nM。
(2)以SS002M91为对照，ELISA评价融合蛋白和CD47结合活性。
将M91和IgG1-FC(序列如SEQ ID NO:71所示)融合得到SS002M91，SS002M91氨基酸序列如SEQ ID NO:72所示。
将靶抗原CD47-His(1μg/ml)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的SS002M91、SG3847L1或SG3847L2，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG二抗(Goat Anti human IgG HRP，Thermo Fisher Scientific)，37℃反应30分钟；PBST洗涤5次；每孔加入100μl TMB (eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析融合蛋白与靶抗原CD47结合能力。
如图3所示，SG3847L1、SG3847L2和SS002M91相比，与靶抗原CD47的结合力相似，SG3847L1、SG3847L2和SS002M91的EC50值分别为0.114nM，0.091nM和0.072nM。
(3)分别以SG003和SS002M91为对照，评价融合蛋白分别与CD38和CD47抗原的结合活性。
使用经工程改造以表达人CD38或人CD47的CHO-S细胞系评价融合蛋白结合细胞表面抗原的能力。收集对数生长期细胞，调整细胞密度到5×10 6个细胞/mL，冰上预冷。含2％FBS预冷的生理盐水将SG003、SS002M91、阴性对照抗体(IgG1)、SG3847L1或SG3847L2稀释成不同浓度。
取100μl细胞，加入等体积前述稀释抗体或融合蛋白，4℃避光反应30min。结束后，洗涤细胞两次。用稀释的PE标记山羊抗人IgG-Fc二抗(PE-Goat anti-human IgG Fc Secondary Antibody，eBioscience)100μL重悬细胞，4℃避光反应30min。反应结束后，洗涤细胞两次。用400μl 1％多聚甲醛重悬细胞。流式细胞仪(BD Calibur)分析融合蛋白与细胞表面CD38或CD47的结合能力。
如图4A和4B所示，“1”、“2、“3”、“4”、“5”、“6”、“7”、“8”和“9”分别是指20μgml -1IgG1、20μgml -1SG003、20μgml -1SS002M91、20μgml -1SG3847L1、10μgml - 1SG3847L1、5μgml -1SG3847L1、20μgml -1SG3847L2、10μgml -1SG3847L2和5μgml -1SG3847L2。结果显示，融合蛋白SG3847L1和SG3847L2能特异性识别细胞表面表达的CD38或CD47抗原。
1.3与两个抗原同时结合的活性分析
(1)参考图5的原理，以抗CD38人源化抗体SG003为对照，ELISA分析融合蛋白SG3847L1和SG3847L2同时结合两抗原CD38和CD47的生物学活性。
将靶抗原CD38-His(1μg/ml)包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的SG003抗体、SG3847L1或SG3847L2融合蛋白，37℃反应1小时；PBST洗涤后，加入生物素标记CD47(Biotin-Fc-CD47)，37℃反应30分钟；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃反应30分钟；PBST洗涤5次；每孔加入100μl TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析融合蛋白与靶抗原CD38、CD47同时结合的能力。
如图6所示，融合蛋白SG3847L1、SG3847L2和SG003抗体相比，可与靶抗原CD38和 CD47同时结合，SG3847L1和SG3847L2的EC50值分别为0.020nM和0.024nM。
(2)参考图7的原理，以SG003和SS002M91为对照，评价融合蛋白SG3847L1、SG3847L2同时结合细胞表面两抗原CD38和CD47的生物学活性。
使用经工程改造以表达人CD38的CHO-S细胞系评价融合蛋白同时结合细胞表面双抗原的能力。收集对数生长期细胞，调整细胞密度到5×10 6个细胞/mL，冰上预冷。含2％FBS预冷的生理盐水将SG003抗体、SS002M91融合蛋白、阴性对照抗体(IgG1)、SG3847L1或SG3847L2稀释。取100μl细胞，加入等体积前述稀释抗体或融合蛋白及CD47-FITC(嘉暄生物)，4℃避光反应30min。结束后，洗涤细胞两次。用稀释的PE标记山羊抗人IgG-Fc二抗(PE-Goat anti-human IgG Fc Secondary Antibody，eBioscience)即Fc-PE 100μL重悬细胞，4℃避光反应30min。反应结束后，洗涤细胞两次。用400μl 1％多聚甲醛重悬细胞。流式细胞仪(BD Calibur)分析融合蛋白与细胞表面CD38和CD47同时结合的能力。
如图8所示，融合蛋白SG3847L1、SG3847L2能同时识别细胞表面的CD38和CD47抗原，并且SG3847L2同时识别双抗原的能力更高。
1.4杀伤CD38阳性细胞的ADCC活性分析
首先将实验所需靶细胞(Raji细胞)调整密度至2×10 5个细胞/mL重悬在ADCC缓冲液(无酚红MEM培养液+1％FBS)中，并加入96孔板(50μL/孔)。随后向每孔中加入100μL不同浓度的SG003、SG3847L1和SG3847L2，混匀后在37℃，5％CO 2培养箱中孵育30min。再将实验所需效应细胞NK92MI-CD16a(华博生物)调整密度至1.2×10 6个细胞/mL，加入有靶细胞的孔中，使得靶细胞：效应细胞＝1:6。混匀后在37℃，5％CO 2培养箱中孵育6h后，去掉96孔板内原液100μL/孔，加入LDH检测试剂盒(Cytotoxicity Detection Kit，Roche)中LDH反应混合物100μL/孔，37℃反应10min。再加入终止液50μL/孔，轻微混匀。酶标仪测定OD492nm，同时测定OD650nm作为背景值。实验中同时设置如下对照组，其中对照1为ADCC缓冲液，对照2为靶细胞+ADCC缓冲液，对照3为靶细胞+裂解液+ADCC缓冲液，对照4为靶细胞+效应细胞+ADCC缓冲液。特异性杀伤率％＝((实验组-对照4)/(对照3-对照2))×100％。量效曲线采用GraphPad Prism Version 5进行数据分析。
如图9所示，使用Raji靶细胞和NK92MI-CD16a效应细胞检测时，融合蛋白SG3847L1、SG3847L2显示出了更好的ADCC作用。SG003、SG3847L1和SG3847L2的EC50值分别为0.067nM、0.014nM和0.013nM。
1.5阻断CD47蛋白与SIRPa相互作用活性分析
以SS002M91为对照，评价融合蛋白SG3847L1、SG3847L2阻断CD47蛋白与SIRPa相互作用的生物学活性。
将SIRPα-His包被酶联板，1μg/ml，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；使用10％的胎牛血分别梯度稀释SS002M91、SG3847L1或SG3847L2，并在样品中加入Biotin-Fc-CD47至终浓度2μg/ml，37℃预孵育30min，作为一抗；PBST洗涤酶联板后，加入一抗，37℃孵育1小时；PBST洗5遍，加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃孵育30分钟；PBST洗5遍，每孔加入100μl TMB(eBioscience)，室温(20±5℃)避光放置1-5min；每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析SS002M91、SG3847L1、SG3847L2对CD47/SIRPα的阻断作用。
从图10中可以看出，SG3847L1、SG3847L2与SS002M91一样，可以竞争性阻断CD47与其配体SIRPα的结合。SG3847L1的IC50值为46.8nM，SG3847L2的IC50值为78.8nM，SS002M91的IC50值为45.7nM。
实施例2 采用不同靶点抗体与人SIRPα变体1的突变体组合
参照如图1的融合蛋白结构以及实施例1的结果，选用抗AXL抗体(C6G12)和抗Trop2(SG701)抗体为例，研究不同靶点抗体融合蛋白生物学活性的影响，采用的连接子为连接子2。
抗体C6G12的LCDR1-3的氨基酸序列分别如SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24所示；VL的氨基酸序列如SEQ ID NO:28所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27所示；VH的氨基酸序列如SEQ ID NO:29所示；轻链的氨基酸序列如SEQ ID NO:32所示；重链的氨基酸序列如SEQ ID NO:34所示。
抗体SG701的LCDR1-3的氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:39所示；VL的氨基酸序列如SEQ ID NO:42所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示；VH的氨基酸序列如SEQ ID NO:43所示；轻链的氨基酸序列如SEQ ID NO:46所示；重链的氨基酸序列如SEQ ID NO:48所示。
以抗AXL抗体(C6G12)、SIRPα变体1的突变体M91和连接子2(SEQ ID:NO 74)制备融合蛋白，从N端到C端，依次连接C6G12、连接子和2个M91，其中2个M91的N端分别和C6G12的重链的C端连接，得到的融合蛋白命名为SGAXL47。SGAXL47的所述第二多肽链和所述第一多肽链的氨基酸序列分别如SEQ ID NO:77和SEQ ID NO:32所示。
以抗Trop抗体(SG701)、SIRPα变体1的突变体M91和连接子2(SEQ ID:NO 74)制备融合蛋白，从N端到C端，依次连接SG701、连接子和2个M91，其中2个M91的N端分别和SG701的重链的C端连接，得到的融合蛋白命名为SGTrop247。SGTrop247的所述第二多肽链和所述第一多肽链的氨基酸序列分别如SEQ ID NO:78和SEQ ID NO:46所示。
采用常规分子生物学技术，将SGAXL47和SGTrop247融合蛋白的表达基因构建真核表达载体，转染CHO-S细胞后，收获上清，经过Protein A亲和纯化，获得目的蛋白，合理评价其生物学活性。
2.1与双抗原结合的活性分析
(1)以抗不同抗原的人源化抗体为对照，ELISA评价相应融合蛋白和相关抗原的结合活性。
将靶抗原包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的抗体或融合蛋白，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG二抗(Goat Anti human IgG HRP，Thermo Fisher Scientific)，37℃反应30分钟；PBST洗涤5次；每孔加入100μl TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析融合蛋白与相关靶抗原结合能力。
如图11A所示，SGAXL47和C6G12相比，对AXL抗原的结合能力接近。如图11B所示，SGTrop247和SG701相比，对Trop2抗原的结合能力接近。说明在融合蛋白中采用不同靶点的抗体，并不影响该抗体与Trop2抗原的结合力。
(2)以SS002M91为对照，ELISA评价融合蛋白和CD47的结合活性。
将靶抗原CD47包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的融合蛋白SS002M91或融合蛋白，37℃反应1小时；PBST洗涤后，加入辣根过氧化物酶标记的羊抗人IgG二抗(Goat Anti human IgG HRP，Thermo Fisher Scientific)，37℃反应30分钟；PBST洗涤5次；每孔加入100μl TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析融合蛋白与CD47结合能力。
如图12A所示，SGAXL47和SS002M91相比，对CD47抗原的结合能力接近。如图12B所示，SGTrop247和SS002M91相比，对CD47抗原的结合能力接近。说明在融合蛋白中采用不同靶点的抗体，并不影响该抗体与其CD47抗原的结合力。
2.2.同时结合两个抗原的活性分析
以抗不同抗原的人源化抗体为对照，ELISA分析融合蛋白同时结合双抗原的生物学活性。
将靶抗原包被ELISA板条，4℃过夜；PBST洗涤后，加入10％的胎牛血清，37℃封闭1小时；加入不同浓度的抗体或融合蛋白，37℃反应1小时；PBST洗涤后，加入生物素标记CD47(Biotin-Fc-CD47)，37℃反应30分钟；PBST洗涤5次；加入辣根过氧化物酶标记的亲和素(Streptavidin-HRP，嘉暄生物)，37℃反应30分钟；PBST洗涤5次；每孔加入100 μl TMB(eBioscience)，室温(20±5℃)避光放置1～2min；随后每孔加入100μl 2N H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，分析融合蛋白与相关抗原和CD47同时结合的能力。
图13A显示，SGAXL47和C6G12分别对AXL和CD47双抗原的结合情况，图13B显示，SGTrop247和SS002M91分别对Trop2和CD47双抗原的结合情况。图13结果显示融合蛋白替换不同抗体，并不影响其与双抗原同时结合。
实施例3 融合蛋白体内抑制肿瘤活性
CB17SCID小鼠尾静脉接种Raji-Luc细胞建立肿瘤模型，评价融合蛋白SG3847L1抑制肿瘤活性的效果。
选择雌性，5～8周龄的CB17SCID小鼠(购自北京百奥赛图基因生物技术有限公司)进行试验。Raji-Luc细胞为北京百奥赛图基因生物技术有限公司在Raji细胞的基础上，转入荧光素报告基因后获得的稳定细胞系。Raji-Luc细胞复苏培养至所需数量后，收集对数期生长细胞悬浮至2.5×10 7个/mL浓度，以0.2mL/只的数量通过尾静脉接种CB17SCID小鼠，接种后于第0天、第7天用小动物成像仪观察肿瘤生长情况及体重，并于第7天挑选肿瘤成像信号适中的30只小鼠，随机分配到5个组中，每组6只。然后开始对动物进行给药1次，设生理盐水对照组、实验组(SG003，100ug/kg)、实验组(SS002M91,50ug/kg)、联合给药组(SG003+SS002M91,100ug/kg+50ug/kg)、实验组(SG3847L1,120ug/kg)，观察小鼠体重、肿瘤生长情况。
结果显示，SG003抗体、SS002M91融合蛋白以及SG3847L1融合蛋白均可为实验动物所耐受。各组给药动物和生理盐水对照组比较，有明显肿瘤生长抑制效果(参见图14)；从图15可以看出，给药后第七天，SG003抗体治疗组、SS002M91融合蛋白治疗组、SG003抗体和SS002M91融合蛋白联合给药治疗组以及SG3847L1融合蛋白治疗组的平均荧光强度分别为1.60E+07(标准误：1.02E+07)、2.42E+07(标准误：6.70E+06)、4.61E+06(标准误：7.57E+05)、5.97E+05(标准误：1.10E+05)，均显著低于对照组平均荧光强度8.04E+08(标准误1.15E+08)。同时SG3847L1融合蛋白治疗组对肿瘤生长抑制效果显著优于SG003抗体和SS002M91融合蛋白联合用药组。
此外，从图16可以看出，其中图中“G1生理盐水”、“G2SG003”、“G3SS002M91”、“G4SG003+SS002M91”和“G5SG3847L1”分别表示“生理盐水组”、“SG003抗体治疗组”、“SS002M91融合蛋白治疗组”、“SG003和SS002M91联合治疗组”和“SG3847L1融合蛋白治疗组”，生理盐水组的中位生存期是11天，SG003抗体治疗组和SS002M91融合 蛋白治疗组的中位生存期分别是22天和21天，说明SG003和SS002M91显著延长了小鼠生存期。截止分组后23天，SG003和SS002M91联合治疗组以及SG3847L1融合蛋白治疗组的小鼠均未出现死亡。说明，与SG003和SS002M91单独治疗相比，SG003和SS002M91联合治疗组以及SG3847L1单独治疗组均延长了小鼠生存期。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (61)

  1. 融合蛋白，其包含：特异性结合肿瘤相关抗原的第一结合域；以及特异性结合CD47蛋白的第二结合域；其中，所述第二结合域包含人SIRPα变体1的突变体，所述突变体与SEQ ID NO:50所示的序列相比，在第33位至第149位中的一个或多个位置包含氨基酸残基的取代、缺失或添加。
  2. 根据权利要求1所述的融合蛋白，其中所述突变体在选自下组的一个或多个氨基酸残基处包含氨基酸取代：R22、I29、I61、V63、E77、Q82、K83、E84、V93、D95、D96、K98、N100、R107、G109和V132。
  3. 根据权利要求2所述的融合蛋白，其中所述突变体在选自下组的氨基酸残基处包含氨基酸取代：
    (1)I61、V63、E77、E84、V93、L96、K98、N100和V132；
    (2)I61、E77、Q82、K83和E84；
    (3)I61、V63、K83、E84和V132；
    (4)I61、E77、E84、R107和V132；
    (5)I61、V63、E77、K83、E84和N100；
    (6)I61、E77、Q82、K83、E84和R107；
    (7)I61、E77、Q82、E84、V93、L96、N100、R107、G109和V132；
    (8)I61、E77、Q82、K83、E84和V132；
    (9)I61；
    (10)I61、D95、L96、G109和V132；
    (11)I61、D95、L96、K98、G109和V132；
    (12)I61、E77、E84、V93、R107和V132；
    (13)E77、L96、N100、G109和V132；
    (14)I61、V63、Q82、E84、D95、L96、N100和V132；
    (15)I61、E77、Q82、K83、E84、V93、D95、L96、K98、N100和V132；
    (16)I61、E77、Q82、K83、E84和V93；
    (17)I61、V63、E77、K83、E84、D95、L96、K98和N100；
    (18)I61、V63、E77、K83、D95、L96、K98、N100和G109；
    (19)I61、E77、Q82、E84、V93、D95、L96、K98和N100；和，
    (20)I61、V63、E77、Q82和E84。
  4. 根据权利要求1-3中任一项所述的融合蛋白，其中所述突变体包含选自下组的一个或多个氨基酸取代：R22C、I29L、I61L/V/F、V63I、E77I/N/Q/K/H/M/R/N/V/L、Q82S/R/G/N、 K83R、E84K/H/D/R/G、V93L/A、D95H/R/E、D96S/T、K98R、N100G/K/D/E、R107N/S、G109R/H和V132L/R/I/S。
  5. 根据权利要求4所述的融合蛋白，其中所述突变体包含选自下组的氨基酸取代：
    (1)I61L、V63I、E77I、E84K、V93L、L96S、K98R、N100G和V132L；
    (2)I61V、E77N、Q82S、K83R和E84H；
    (3)I61F、V63I、K83R、E84K和V132I；
    (4)I61L、E77Q、E84D、R107N和V132I；
    (5)I61L、V63I、E77K、K83R、E84D和N100G；
    (6)I61V、E77H、Q82R、K83R、E84H和R107S；
    (7)I61L、E77I、Q82G、E84R、V93L、L96T、N100G、R107S、G109R和V132R；
    (8)I61L、E77M、Q82G、K83R、E84D和V132L；
    (9)I61L；
    (10)I61F、D95H、L96S、G109H和V132S；
    (11)I61F、D95H、L96S、K98R、G109H和V132S；
    (12)I61L、E77Q、E84D、V93A、R107N和V132I；
    (13)E77K、L96S、N100K、G109H和V132L；
    (14)I61L、V63I、Q82G、E84G、D95R、L96S、N100D和V132I；
    (15)I61L、E77R、Q82N、K83R、E84G、V93L、D95E、L96T、K98R、N100D和V132L；
    (16)I61V、E77N、Q82S、K83R、E84H和V93A；
    (17)I61V、V63I、E77V、K83R、E84D、D95E、L96T、K98R和N100E；
    (18)I61L、V63I、E77V、K83R、D95E、L96S、K98R、N100D和G109R；
    (19)I61V、E77L、Q82G、E84G、V93L、D95E、L96T、K98R和N100G；和，
    (20)I61L、V63I、E77N、Q82G和E84G。
  6. 根据权利要求1-5中任一项所述的融合蛋白，其中所述突变体包含如SEQ ID NO:51-70中任一项所示的氨基酸序列。
  7. 根据权利要求1-6中任一项所述的融合蛋白，其中所述第一结合域包含抗体或其抗原结合片段或变体。
  8. 根据权利要求7所述的融合蛋白，其中所述抗体选自下组：单克隆抗体、单链抗体、嵌合抗体、人源化抗体和全人源抗体。
  9. 根据权利要求7-8中任一项所述的融合蛋白，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab')2，F(ab)2，dAb，分离的互补决定区CDR，Fv和scFv。
  10. 根据权利要求7-9中任一项所述的融合蛋白，其中所述抗体或其抗原结合片段的所述变体选自下组：
    a)在所述抗体或所述其抗原结合片段中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽；和
    b)与所述抗体或所述其抗原结合片段具有至少90％序列同源性的蛋白质或多肽。
  11. 根据权利要求1-10中任一项所述的融合蛋白，其中所述肿瘤相关抗原包括与非实体瘤和/或实体瘤相关的肿瘤相关抗原。
  12. 根据权利要求1-11中任一项所述的融合蛋白，其中所述肿瘤相关抗原选自以下组：CD38、AXL和Trop2。
  13. 根据权利要求12所述的融合蛋白，其中所述第一结合域包含CD38抗体或其抗原结合片段或变体。
  14. 根据权利要求13所述的融合蛋白，其中所述CD38为人CD38。
  15. 根据权利要求7-14中任一项所述的融合蛋白，其中所述抗体包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6。
  16. 根据权利要求15所述的融合蛋白，其中所述抗体重链或其片段包含重链可变区VH，且所述重链可变区VH包含如下的氨基酸序列：SEQ ID NO:8。
  17. 根据权利要求15-16中任一项所述的融合蛋白，其中所述抗体重链或其片段包含重链恒定区，且所述重链恒定区包含IgG。
  18. 根据权利要求17所述的融合蛋白，其中所述IgG选自下组：IgG1和IgG4。
  19. 根据权利要求15-18中任一项所述的融合蛋白，其中所述抗体重链包含选自下组所示的氨基酸序列中的任一项：SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:19和SEQ ID NO:21。
  20. 根据权利要求7-19中任一项所述的融合蛋白，其中所述抗体包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3。
  21. 根据权利要求20所述的融合蛋白，其中所述抗体轻链或其片段包含轻链可变区VL，且所述轻链可变区VL包含如下的氨基酸序列：SEQ ID NO:7。
  22. 根据权利要求20-21中任一项所述的融合蛋白，其中所述抗体轻链或其片段包含轻链恒定区，其所述轻链恒定区包含Igκ。
  23. 根据权利要求20-22中任一项所述的融合蛋白，其中所述抗体轻链包含选自下组所示 的氨基酸序列中的任一项：SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:18和SEQ ID NO:20。
  24. 根据权利要求12所述的融合蛋白，其中所述第一结合域包含AXL抗体或其抗原结合片段或变体。
  25. 根据权利要求24所述的融合蛋白，其中所述AXL为人AXL。
  26. 根据权利要求24-25中任一项所述的融合蛋白，其中所述抗体包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27。
  27. 根据权利要求26所述的融合蛋白，其中所述抗体重链或其片段包含重链可变区VH，且所述重链可变区VH包含如下的氨基酸序列：SEQ ID NO:29。
  28. 根据权利要求26-27中任一项所述的融合蛋白，其中所述抗体重链或其片段包含重链恒定区，且所述重链恒定区包含IgG。
  29. 根据权利要求28所述的融合蛋白，其中所述IgG选自下组：IgG1和IgG4。
  30. 根据权利要求24-29中任一项所述的融合蛋白，其中所述抗体重链包含如下的氨基酸序列：SEQ ID NO:34。
  31. 根据权利要求24-30中任一项所述的融合蛋白，其中所述抗体包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24。
  32. 根据权利要求31所述的融合蛋白，其中所述抗体轻链或其片段包含轻链可变区VL，且所述轻链可变区VL包含如下的氨基酸序列：SEQ ID NO:28。
  33. 根据权利要求31-32中任一项所述的融合蛋白，其中所述抗体轻链或其片段包含轻链恒定区，其所述轻链恒定区包含Igκ。
  34. 根据权利要求31-33中任一项所述的融合蛋白，其中所述抗体轻链包含如下的氨基酸序列：SEQ ID NO:32。
  35. 根据权利要求12所述的融合蛋白，其中所述第一结合域包含Trop2抗体或其抗原结合片段或变体。
  36. 根据权利要求35所述的融合蛋白，其中所述Trop2为人Trop2。
  37. 根据权利要求35-36中任一项所述的融合蛋白，其中所述抗体包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1-3，所述HCDR1-3的氨基酸序列依次为SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41。
  38. 根据权利要求37所述的融合蛋白，其中所述抗体重链或其片段包含重链可变区VH， 且所述重链可变区VH包含如下的氨基酸序列：SEQ ID NO:43。
  39. 根据权利要求37-38中任一项所述的融合蛋白，其中所述抗体重链或其片段包含重链恒定区，且所述重链恒定区包含IgG。
  40. 根据权利要求39所述的融合蛋白，其中所述IgG选自下组：IgG1和IgG4。
  41. 根据权利要求37-40中任一项所述的融合蛋白，其中所述抗体重链包含如下的氨基酸序列：SEQ ID NO:48。
  42. 根据权利要求35-41中任一项所述的融合蛋白，其中所述抗体包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1-3，所述LCDR1-3的氨基酸序列依次为SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38。
  43. 根据权利要求42所述的融合蛋白，其中所述抗体轻链或其片段包含轻链可变区VL，且所述轻链可变区VL包含如下的氨基酸序列：SEQ ID NO:42。
  44. 根据权利要求42-43中任一项所述的融合蛋白，其中所述抗体轻链或其片段包含轻链恒定区，其所述轻链恒定区包含Igκ。
  45. 根据权利要求42-44中任一项所述的融合蛋白，其中所述抗体轻链包含如下的氨基酸序列：SEQ ID NO:46。
  46. 根据权利要求1-45中任一项所述的融合蛋白，其中所述第一结合域位于所述第二结合域的N端。
  47. 根据权利要求1-46中任一项所述的融合蛋白，其中所述融合蛋白还包含连接子，所述连接子位于所述第一结合域的C端且位于所述第二结合域的N端。
  48. 根据权利要求47所述的融合蛋白，其中所述连接子包含如SEQ ID NO:73-74中任一项所示的氨基酸序列。
  49. 根据权利要求1-48中任一项所述的融合蛋白，其包含至少2个所述第二结合域。
  50. 根据权利要求49所述的融合蛋白，其中所述每个所述第二结合域分别位于所述第一结合域的C端。
  51. 免疫缀合物，其包含根据权利要求1-50中任一项所述的融合蛋白。
  52. 一个或多个分离的核酸分子，其编码根据权利要求1-50中任一项所述的融合蛋白或者根据权利要求51所述的免疫缀合物。
  53. 一个或多个载体，其包含权利要求52所述的核酸分子。
  54. 组合物，其包含根据权利要求1-50中任一项所述的融合蛋白，根据权利要求51所述的免疫缀合物，或根据权利要求52所述的核酸分子，以及任选地药学上可接受的赋形剂。
  55. 细胞，其包含权利要求1-50中任一项所述的融合蛋白，权利要求51所述的免疫缀合 物，权利要求52所述的核酸分子，或权利要求53所述的载体。
  56. 制备根据权利要求1-50中任一项所述的融合蛋白的方法，其包括在使得所述融合蛋白能够表达的条件下培养根据权利要求55所述的细胞。
  57. 权利要求1-50中任一项所述的融合蛋白，权利要求51所述的免疫缀合物，权利要求52所述的核酸分子，权利要求53所述的载体，权利要求54所述的组合物，或权利要求55所述的细胞在制备药物中的用途，其中所述药物用于治疗肿瘤或自免疫疾病。
  58. 根据权利要求57所述的用途，其中所述肿瘤包括非实体瘤和实体瘤。
  59. 根据权利要求57所述的用途，其中所述肿瘤包括多发性骨髓瘤、白血病、非霍奇金淋巴瘤、霍奇金淋巴瘤、神经胶质瘤、生殖细胞瘤、肉瘤、见皮瘤、胎盘瘤、脑癌、骨癌、皮肤癌、鼻咽癌、肺癌、口腔癌、食道癌、胃癌、肝癌、胰腺癌、前列腺癌、肠癌、乳腺癌、宫颈癌、卵巢癌和睾丸癌。
  60. 根据权利要求57所述的用途，其中所述自免疫疾病包括慢性淋巴性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、慢性溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天皰疮、类天皰疮、原发性胆汁性肝硬变、多发性脑脊髓硬化症、急性特发性多神经炎、系统性红斑狼疮、类风湿性关节炎、硬皮病和结节性多动脉炎。
  61. 阻断CD47蛋白与SIRPα相互作用的方法，其包括向有需要的受试者施用有效量的权利要求1-50中任一项所述的融合蛋白，权利要求51所述的免疫缀合物，权利要求52所述的核酸分子，权利要求53所述的载体，权利要求54所述的组合物，或权利要求55所述的细胞。
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