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WO2020200256A1 - 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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WO2020200256A1
WO2020200256A1 PCT/CN2020/082861 CN2020082861W WO2020200256A1 WO 2020200256 A1 WO2020200256 A1 WO 2020200256A1 CN 2020082861 W CN2020082861 W CN 2020082861W WO 2020200256 A1 WO2020200256 A1 WO 2020200256A1
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fusion protein
seq
amino acid
cancer
antibody
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PCT/CN2020/082861
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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
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Priority to BR112021019570A priority Critical patent/BR112021019570A2/pt
Priority to AU2020250613A priority patent/AU2020250613A1/en
Priority to SG11202110400QA priority patent/SG11202110400QA/en
Priority to CN202080023723.7A priority patent/CN113631582B/zh
Priority to JP2021556959A priority patent/JP2022527705A/ja
Priority to CA3131075A priority patent/CA3131075A1/en
Priority to US17/599,733 priority patent/US20220251154A1/en
Priority to KR1020217030645A priority patent/KR20210146916A/ko
Priority to EP20782966.4A priority patent/EP3950720A4/en
Publication of WO2020200256A1 publication Critical patent/WO2020200256A1/zh

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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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Definitions

  • This application relates to the field of biomedicine, specifically to a multispecific fusion protein, and also to its use in the treatment of tumors and/or autoimmune diseases.
  • Programmed death protein 1 (programmed death 1, PD-1) antibody is an immunotherapy.
  • PD-1 is expressed in activated T cells, B cells and myeloid cells, and it has two ligands, namely programmed death ligand 1, PD-L1 and PD-L2.
  • PD-1 and/or PD-L1 inhibitors can specifically bind to PD-L1 on tumor cells to achieve anti-tumor effects, but their clinical response rate is low, and they can also bring some side effects, such as pneumonia and colitis , Hepatitis, etc.
  • CD47 protein is a transmembrane glycoprotein, 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 phagocytosis of tumor cells, which is regarded as a mechanism for tumors to evade immune surveillance. Blocking the interaction between CD47 protein and SIRP ⁇ can inhibit tumor growth.
  • the application provides a fusion protein comprising a first binding domain that specifically binds PD-L1 and a second binding domain that specifically binds CD47 protein.
  • the application also provides immunoconjugates containing 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 for 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 to CD47 protein and PD-L1 at the same time; 2) can specifically block The interaction between CD47 protein and SIRP ⁇ ; 3) can specifically block the interaction between PD-1 and PD-L1; 4) 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 PD-L1; and a second binding domain that specifically binds to CD47 protein; wherein the second binding domain comprises A mutant of human SIRP ⁇ variant 1, which, compared with the sequence shown in SEQ ID NO: 29, contains substitutions, deletions, or substitutions of amino acid residues at one or more positions from 33 to 149 Add to.
  • the mutant contains 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, L96 , K98, N100, R107, G109 and V132.
  • the mutant contains 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 contains 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, L96S/T, K98R, N100G/K/D/E, R107N/S, G109R/H and V132L/R/I/S.
  • 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, L96S/T, K98R, N100G/K/D
  • 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, D
  • the mutant comprises an amino acid sequence as shown in any one of SEQ ID NO: 30-49.
  • 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 region CDR, Fv and scFv.
  • the PD-L1 is human PD-L1.
  • the antibody comprises an antibody heavy chain or a fragment thereof, the antibody heavy chain or a fragment thereof comprises HCDR1-3, and the HCDR1 comprises an amino acid sequence shown in any one of the following group: SEQ ID NO: 4 and SEQ ID NO: 18.
  • the HCDR2 includes an amino acid sequence shown in any one of the following groups: SEQ ID NO: 5 and SEQ ID NO: 19.
  • the HCDR3 includes an amino acid sequence shown in any one of the following groups: SEQ ID NO: 6 and SEQ ID NO: 20.
  • 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 shown in any one of the following groups: SEQ ID NO: 8 and SEQ ID NO: 22.
  • 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 shown in any one of the following groups: SEQ ID NO: 13 and SEQ ID NO: 27.
  • the antibody comprises an antibody light chain or a fragment thereof, the antibody light chain or a fragment thereof comprises LCDR1-3, and the LCDR1 comprises an amino acid sequence shown in any one of the following group: SEQ ID NO: 1 and SEQ ID NO: 15.
  • the LCDR2 includes an amino acid sequence shown in any one of the following groups: SEQ ID NO: 2 and SEQ ID NO: 16.
  • the LCDR3 includes an amino acid sequence shown in any one of the following groups: SEQ ID NO: 3 and SEQ ID NO: 17.
  • the antibody light chain or a fragment thereof includes a light chain variable region VL, and the light chain variable region VL includes an amino acid sequence shown in any one of the following groups: SEQ ID NO: 7 and SEQ ID NO: 21.
  • the antibody light chain or fragment thereof comprises a light chain constant region, and the light chain constant region comprises Ig ⁇ .
  • the antibody light chain comprises an amino acid sequence shown in any one of the following groups: SEQ ID NO: 11 and SEQ ID NO: 25.
  • 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 includes the amino acid sequence shown in SEQ ID NO: 52.
  • 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.
  • this application provides an immunoconjugate comprising the fusion protein.
  • the present application provides one or more isolated nucleic acid molecules, which encode the fusion protein or the immunoconjugate.
  • the present application provides one or more vectors, which comprise 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.
  • this application provides a cell comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, or the vector.
  • the present application provides a method for preparing the fusion protein, which includes culturing the cell under conditions that enable the expression of the fusion protein.
  • the 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 medicine , Wherein the drug is used to treat tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the solid tumors and non-solid tumors include multiple myeloma, leukemia, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, glioma, germ cell tumor, sarcoma, dermatoma , Placental tumor, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, bowel cancer, breast cancer, cervical cancer, ovarian cancer and testicular cancer, Upper frontal sinus tumor, hypopharyngeal cancer, olfactory blastoma, tongue cancer, gum cancer, ampullary cancer, colon cancer, rectal cancer, kidney cancer, ureter cancer, bladder cancer, penile cancer, fallopian tube cancer, eyelid cancer, oviduct cancer Cell tumor.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the composition, or the cell, which treats tumors.
  • the present application provides a method for blocking the interaction between PD-L1 protein and PD-1, which comprises administering an effective amount of the fusion protein to a subject in need, and the immunoconjugate Thing, said nucleic acid molecule, said vector, said composition, or said cell.
  • the present application provides a method for blocking the interaction between CD47 protein and SIRP ⁇ , which comprises administering to a subject in need an effective amount of the fusion protein, the immunoconjugate, the The nucleic acid molecule, the vector, 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 to a subject in need an effective amount of the fusion protein, the immunoconjugate, The nucleic acid molecule, the vector, the composition, or the cell.
  • the present application provides a method for preventing or treating tumors in a subject, which comprises administering to a subject in need an effective amount of the fusion protein, the immunoconjugate, and The nucleic acid molecule, the vector, the composition, or the cell.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the carrier, the composition, or the cell, which prevents or treats Tumor in the subject.
  • Figure 1 shows an example of the structure of the fusion protein described in this application.
  • Figure 2-3 shows the binding ability of the fusion protein described in this application to PD-L1.
  • Figure 4 shows the binding ability of the fusion protein described in this application to CD47.
  • Figure 5 shows the ability of the fusion protein described in this application to simultaneously bind to PD-L1 and CD47.
  • Figure 6 shows that the fusion protein described in this application competitively blocks the binding of CD47 to its ligand SIRP ⁇ .
  • Figures 7-8 show that the fusion protein described in this application competitively blocks the binding of PD-1 and PD-L1.
  • Figures 9-10 show the use of the fusion protein described in this application to inhibit the growth of tumor volume in a mouse human lymphoma tumor model.
  • fusion protein generally refers to a protein obtained by fusing two or more proteins or polypeptides.
  • the fusion protein can be artificially prepared by recombinant DNA technology.
  • the genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or fusion nucleic acid molecule, and the fusion gene or fusion nucleic acid molecule can encode the fusion protein.
  • the translation of the fusion gene can produce a single polypeptide, which can have the properties of at least one or even each of the two or more proteins or polypeptides before the fusion.
  • the term "specific binding” generally refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen producing 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, Wherein KD refers to the ratio of dissociation rate to binding rate (koff/kon), which can be determined by a method familiar to those skilled in the art.
  • binding domain generally refers to a domain that can specifically bind and/or recognize a specific epitope on a target (eg, an antigen).
  • domain generally refers to regions of tight globular structures that are clearly separated in the structure of protein subunits.
  • a polypeptide chain can first have a regular secondary structure formed by adjacent amino acid residues in certain regions, and then can be assembled from adjacent secondary structure fragments to form a super secondary structure, on this basis
  • the polypeptide chain can be folded into a tertiary structure that is approximately spherical.
  • the polypeptide chain can often be composed of two or more spatially distinct, relatively independent regional structures associated to form a tertiary structure, this relatively independent regional structure It can be called a domain.
  • first binding domain and “second binding domain” may only be used for descriptive distinction.
  • CD47 protein generally refers to Integrin-Associated Protein (IAP), which is a multi-transmembrane receptor belonging to the immunoglobulin superfamily.
  • IAP Integrin-Associated Protein
  • CD47 protein can bind to membrane integrins, and bind to its ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRP ⁇ ).
  • TSP-1 thrombospondin-1
  • SIRP ⁇ signal-regulatory protein alpha
  • CD47 protein is widely expressed on the surface of cell membranes.
  • the CD47 protein may include any variant, isotype, and species homologue 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 CD47 gene.
  • SIRP ⁇ generally refers to a regulatory membrane glycoprotein from the SIRP family, which can act as a ligand for the CD47 protein.
  • the SIRP ⁇ may include human SIRP ⁇ .
  • the SIRP ⁇ variant 2 is different from the SIRP ⁇ variant 1 by 13 amino acids, and its amino acid sequence is listed as CAA71403.1 in GenBank.
  • SIRP ⁇ variant 1 usually refers to the SIRP ⁇ protein whose amino acid sequence is listed as NCBI RefSeq NP_542970.1 (residues 31-504 constitute the mature form). At this time, the amino acid sequence of SIRP ⁇ variant 1 is as SEQ ID NO: 29.
  • antibody generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments.
  • immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as countless immunoglobulin variable region genes.
  • light chains can be classified as kappa or lambda, which can define immunoglobulin types: Ig ⁇ and Ig ⁇ , respectively.
  • Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE.
  • an antibody may have a structural unit comprising tetramers, and each tetramer may be composed of two pairs of identical polypeptide chains, each pair having a "light” chain (about 25kD) and a "heavy chain” (about 50-70kD). ), the N-terminus of each member can 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 variable region regions of the light chain and heavy chain, respectively.
  • Antibodies can exist as whole immunoglobulins or as many well-characterized fragments generated 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 basically retains the ability to bind the same antigen (for example, PD-L1) to which the antibody binds, and can be compared with the whole Long antibodies compete for specific binding to the antigen.
  • PD-L1 antigen-binding fragment
  • antigen-binding fragments include Fab, Fab', F(ab')2, (Fab)2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments , Single-chain antibodies (for example, scFv), chimeric antibodies, diabodies (diabodies) and such polypeptides, which comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Technology obtains the antigen-binding fragment of the antibody from a given antibody, and specifically screens the antigen-binding fragment of the antibody in the same way as for the intact antibody.
  • Pepsin can digest the antibody below the disulfide bond in the hinge region to produce F(ab')2.
  • Fab generally refers to an antibody fragment composed of VL, VH, CL and CH1 domains.
  • Fab' generally refers to an antibody fragment having several additional residues at the carboxyl terminal of the CH1 domain compared to the Fab fragment.
  • Fab' may include one or more cysteines from the hinge region of an 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). This position is due to the spatial structure of the three hypervariable regions (HVR). Forms precise complementarity with antigenic determinants, so the hypervariable region is also called complementarity determining region.
  • Fv fragment generally refers to an antibody fragment composed of the VL and VH domains of a single arm of an antibody.
  • scFv generally refers to a molecule formed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody through 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 contained in the group may be the same except for possible naturally occurring mutations in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody directed against a single determinant on the antigen is not interpreted as requiring any special method. Produce antibodies.
  • the monoclonal antibody can be prepared by hybridoma technology or by using recombinant DNA methods to produce monoclonal antibodies in bacteria, eukaryotic animals or plant cells, or can 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 class, and The rest of the chain is homologous to the corresponding sequence in another species.
  • the variable regions of the light chain and the heavy chain are derived from the variable region of an antibody from one animal species (such as mouse, rat, etc.), while the constant part is homologous to the sequence of an antibody from another species (such as human) .
  • non-human B cells or hybridoma cells can be used to generate variable regions, and 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 possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody whose constant region is of non-human origin.
  • humanized antibody generally refers to reducing the immunogenicity of antibodies, immunoglobulin binding proteins, and polypeptides derived from non-human species (such as mice or rats) to humans, while still retaining A modified antibody with the antigen-binding properties of the original antibody.
  • genetic engineering techniques can be used to prepare humanized antibodies, CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof can be used; including “reshaping” (Verhoeyen, et al.) .,1988 Science 239:1534-1536; Riechmann,et al.,1988 Nature 332:323-337; Tempest,et al.,Bio/Technol 1991 9:266-271), "hyperchimerization” (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) And “ ⁇ ” (veneering), (Mark,et al.,”Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.”
  • human antibody generally refers to an antibody obtained by expressing a gene encoding a human antibody in a genetically engineered antibody gene-deficient animal.
  • human antibody genes can be transgenic or transchromosome technology to transfer all the genes encoding human antibodies to genetically engineered animals with antibody gene deletions, so that the animals express human antibodies
  • the protein, polypeptide, and/or amino acid sequence involved in this application should also be understood to include at least the following range: a variant or homologue that has the same or similar functions as the protein or polypeptide.
  • the variant may be a substitution, deletion, or addition of one or more in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the PD-L1 protein).
  • a protein or polypeptide with multiple amino acids may comprise at least one, such as 1-30, 1-20, or 1-10, and another example, 1, 2, 3, 4, or 5 amino acid substitutions.
  • the functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (for example, substitution, deletion, or addition).
  • the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide before the change.
  • the substitution may be a conservative substitution.
  • the homologue may be at least about 85% (for example, the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or a fragment thereof that specifically binds to the PD-L1 protein)).
  • the polypeptide for example, an antibody or a fragment thereof that specifically binds to the PD-L1 protein.
  • the homologue may be at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more Proteins or polypeptides of sequence homology.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology” can be calculated in the following way: the two sequences to be aligned are compared in the comparison window, and it is determined that the same nucleic acid base (for example, A, T, C, G, I) exists in the two sequences.
  • the same amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • the sequence homology percentage e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • the alignment to determine the percent sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • FASTA and BLAST For a description of the FASTA algorithm, see WRPearson and DJ Lipman's “Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; And DJ Lipman and WRPearson's "Fast and Sensitive Protein Similarity Search", Science, 227:1435-1441, 1989.
  • BLAST algorithm see "A Basic Local Alignment Search Tool” by S. Altschul, W. Gish, W. Miller, EW Myers, and D. Lipman, Journal of Molecular Biology, 215: 403-410 , 1990.
  • the term "PD-L1" usually refers to the ligand of programmed death-1 (PD-1) protein, which may also be referred to as CD274, B7-H or B7H1.
  • PD-1 negatively regulates T cell antigen receptor signaling by interacting with specific ligands (PD-L).
  • the PD-L1 can be a prognostic indicator of various tumors.
  • the PD-L1 may be human PD-L1.
  • the Gene ID of the human PD-L1 in GenBank is 29126.
  • PD-1 generally refers to a member of the synuclein family, which may also be referred to as NACP, PARK1 or PARK4.
  • the PD1 may be human PD1.
  • the Gene ID of the human PD1 in GenBank is 6622.
  • N-terminal generally refers to the end of a polypeptide chain in which an amino acid residue carries a free amino group.
  • C-terminal generally refers to the end of a polypeptide chain in which the amino acid residue carries a free carboxyl group.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • the term “immunoconjugate” generally refers to a polypeptide molecule with immune function conjugated with one or more heterologous molecules (including but not limited to cytotoxin).
  • heterologous molecules including but not limited to cytotoxin
  • conjugation and connection and “fusion” are used interchangeably in this application, and generally refer to the joining together of two or more chemical elements, sequences or components, for example by Including chemical conjugation or recombinant means.
  • the heterologous molecule may be a cytotoxin, a chemotherapeutic drug and the like.
  • the fusion protein described in this application can be conjugated to one or more heterologous molecules (for example, cytotoxin) to obtain the immunoconjugate.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed.
  • the vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • the vector includes: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage And animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papilloma viruses such as papilloma viruses
  • papillary polyoma vacuoles Virus 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 also contain an origin of replication site.
  • the carrier may also include components
  • tumors generally refers to a neoplasm formed by the proliferation of local tissue cells in a mammalian body (for example, cells or their components) under the action of various tumorigenic factors.
  • tumors may include solid tumors and non-solid tumors.
  • Solid tumors can include glioma, germ cell tumor, sarcoma, dermatoma, placental tumor, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, stomach 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's lymphoma, and Hodgkin's lymphoma.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 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 comprise a first binding domain and a second binding domain.
  • the first binding domain can specifically bind to PD-L1; the second binding domain can specifically bind to CD47 protein, and the second binding domain can comprise a mutant of human SIRP ⁇ variant 1, which is similar to SEQ.
  • one or more of the 33 to 149 (for example, 1-2, 1-3, 1-4, 1-5, 1- 6, 1-7, 1-8, 1-9, 1-10 or more) positions include substitution, deletion or addition of amino acid residues.
  • the fusion protein described in this application can specifically bind to tumor-associated antigen and CD47 protein at the same time, thereby playing a role in treating tumors and/or autoimmune diseases.
  • first binding domain generally refers to a domain that can specifically bind PD-L1.
  • second binding domain generally refers to a domain that can specifically bind to CD47 protein.
  • 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 containing amino acid substitutions: R22, I29, I61, V63, E77, Q82, K83, E84, V93, D95, L96, K98, N100, R107, G109 and V132.
  • amino acid substitution Xn refers to an amino acid substitution at residue X at position n in the amino acid sequence shown in SEQ ID NO: 29, 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 at position 61 in the amino acid sequence shown in SEQ ID NO:29.
  • the amino acid substitutions can be non-conservative substitutions.
  • the non-conservative substitution may include changing the amino acid residues in the target protein or polypeptide in a non-conservative manner, for example, changing amino acid residues with a certain side chain size or certain characteristics (for example, hydrophilicity) to have different Amino acid residues of side chain size or different characteristics (e.g., hydrophobicity).
  • the amino acid substitutions can also be conservative substitutions.
  • the conservative substitution may include changing the amino acid residues in the target protein or polypeptide in a conservative manner, for example, changing amino acid residues with a certain side chain size or a certain characteristic (for example, hydrophilicity) to have the same or similar Side chain size or amino acid residues of the same or similar characteristics (for example, still hydrophilic).
  • Such conservative substitutions usually do not have a great impact on the structure or function of the resulting protein.
  • the amino acid sequence variant as the fusion protein or fragment thereof may include a variant that does not significantly change the protein structure or function (for example, a mutant that blocks CD47 and human SIRP ⁇ variant 1 that specifically binds to the CD47 protein) Conservative amino acid substitutions.
  • amino acid groups with non-polar side chains alanine, valine, leucine, Isoleucine, proline, phenylalanine, tryptophan and methionine.
  • a group of uncharged amino acids with polar side chains glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • a group of negatively charged amino acids with polar side chains aspartic acid and glutamic acid.
  • Positively charged basic amino acids lysine, arginine and histidine.
  • Amino acids with phenyl 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 V
  • the mutant may comprise one or more selected from the following 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, L96S/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/V/F, V63I, E77I/N/Q/K/H/M/R /N/
  • amino acid substitution "XnY/Z” means that the residue X at position n in the amino acid sequence shown in SEQ ID NO: 29 is substituted with amino acid residue Y or amino acid residue Z, where n is positive Integers, X, Y, and Z are each independently an abbreviation for any amino acid residue, and X is different from Y or Z.
  • amino acid substitution "I61L/V/F” means that residue I at position 61 in the amino acid sequence shown in SEQ ID NO: 29 is substituted with amino acid residue L, V or F.
  • the mutant may comprise amino acid substitutions 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, D
  • 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 any one of SEQ ID NOs: 30-49.
  • 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: 41.
  • the first binding domain may comprise an antibody or an 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 region CDR, Fv and scFv.
  • the antibodies or antigen-binding fragments described in this application can kill tumor cells and/or inhibit tumor growth by specifically binding to PD-L1 protein.
  • the tumor may include a PD-L1 positive tumor.
  • the PD-L1 positive tumor can be selected from the following group: gastric cancer, breast cancer, cervical cancer, lung cancer, head and neck tumor, melanoma, glioma, lymphoepithelioma, esophageal cancer or colorectal cancer.
  • the antibody and its antigen-binding fragment can kill gastric cancer, breast cancer, cervical cancer, lung cancer, head and neck tumors, melanoma, glioma, lymphoepithelioma, esophageal cancer or colorectal cancer cells or Inhibit the growth of stomach cancer, breast cancer, cervical cancer, lung cancer, head and neck cancer, melanoma, glioma, lymphoepithelioma, esophageal cancer or colorectal cancer.
  • the PD-L1 protein described in this application may be human PD-L1 protein or functional fragments thereof.
  • the PD-L1 protein may not be a mouse PD-L1 protein, or may not be a rat PD-L1 protein.
  • the antibodies and antigen-binding fragments described in this application do not substantially bind to mouse PD-L1 protein or rat PD-L1 protein.
  • the antibodies and antigen-binding fragments described in this application can compete with the reference antibody for binding to the PD-L1 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 LCDR1 may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 1 and SEQ ID NO: 15; the LCDR2 It may include the amino acid sequence shown in any one of the following group: SEQ ID NO: 2 and SEQ ID NO: 16; the LCDR3 may include the amino acid sequence shown in any one of the following group: SEQ ID NO: 3 and SEQ ID NO : 17.
  • the heavy chain variable region of the reference antibody may include HCDR1-3, and the HCDR1 may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 4 and SEQ ID NO: 18; the HCDR2 may include The amino acid sequence shown in any one of the following group: SEQ ID NO: 5 and SEQ ID NO: 19; the HCDR3 may include the amino acid sequence shown in any one of the following group: SEQ ID NO: 6 and SEQ ID NO: 20 .
  • the amino acid sequence of the light chain variable region of the reference antibody may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 7 and SEQ ID NO: 21, and the heavy chain of the reference antibody
  • the amino acid sequence of the variable region may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 8 and SEQ ID NO: 22.
  • the light chain of the reference antibody may include the amino acid sequence shown in any one of the following groups: SEQ ID NO: 11 and SEQ ID NO: 25; and the heavy chain of the reference antibody may include any of the following groups.
  • the light chain of the reference antibody may include the amino acid sequence as shown in SEQ ID NO: 11 and the heavy chain of the reference antibody may include the amino acid sequence as shown in SEQ ID NO: 13.
  • the light chain of the reference antibody may include the amino acid sequence as shown in SEQ ID NO: 25 and the heavy chain of the reference antibody may include the amino acid sequence as shown in SEQ ID NO: 27.
  • the antibodies and antigen-binding fragments thereof described in the present application may comprise antibody light chains or fragments thereof.
  • the antibody light chain or fragment thereof may include an Ig ⁇ constant region, for example, may include 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 antibody light chain or a fragment thereof may include LCDR1, and the LCDR1 may include the following amino acid sequence: SEQ ID NO: 15.
  • the antibody light chain or a fragment thereof may include LCDR2, and the LCDR2 may include the following amino acid sequence: SEQ ID NO: 16.
  • the antibody light chain or a fragment thereof may include LCDR3, and the LCDR3 may include the following amino acid sequence: SEQ ID NO: 17.
  • the light chain of the antibody described in the present application or a fragment thereof 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 amino acid sequence of the antibody light chain or a fragment thereof may be: SEQ ID NO: 11.
  • the amino acid sequence of the light chain variable region VL may be: SEQ ID NO:21.
  • the amino acid sequence of the antibody light chain or a fragment thereof may be: SEQ ID NO: 25.
  • the antibodies or antigen-binding fragments thereof described in the present application may comprise antibody heavy chains or fragments thereof.
  • the antibody heavy chain or fragment thereof also includes a human constant region.
  • the human constant region may include a human IgG constant region.
  • the IgG constant region may comprise a human IgG1 constant region or IgG4.
  • the antibody heavy chain or a fragment thereof may include HCDR1, and the HCDR1 may include the following amino acid sequence: SEQ ID NO: 4.
  • the antibody heavy chain or fragments 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 HCDR1, and the HCDR1 may include the following amino acid sequence: SEQ ID NO: 18.
  • the antibody heavy chain or fragments thereof may include HCDR2, and the HCDR2 may include the following amino acid sequence: SEQ ID NO 19.
  • the antibody heavy chain or a fragment thereof may include HCDR3, and the HCDR3 may include the following amino acid sequence: SEQ ID NO: 20.
  • 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 include the following amino acid sequence: SEQ ID NO: 13.
  • the heavy chain variable region VH may include the following amino acid sequence: SEQ ID NO: 22.
  • the antibody heavy chain may include the following amino acid sequence: SEQ ID NO: 27.
  • the amino acid sequence of the light chain of the antibody or antigen-binding fragment thereof described in this application includes SEQ ID NO: 11; and the amino acid sequence of the 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: 25; and the amino acid sequence of the heavy chain includes SEQ ID NO: 27.
  • the amino acid sequence of LCDR1 in the antibody or antigen-binding fragment thereof described in the present application 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; and the amino acid sequence of HCDR3 may include SEQ ID NO: 6.
  • the antibody or antigen-binding fragment thereof may include antibody SG1201 or an antibody having the same LCDR1-3 and HCDR1-3.
  • the light chain of the antibody or antigen-binding fragment thereof described in the present application 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 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 antibody SG1201 or an antibody having the same light chain variable region and heavy chain variable region.
  • the antibody or antigen-binding fragment thereof described in the present application may comprise 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 antibody SG1201 or have the same light chain and heavy chain amino acid sequences.
  • the antibody described in this application may be SG1201.
  • the amino acid sequence of the LCDR1-3 of the antibody SG1201 is shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the amino acid sequence of VL is shown in SEQ ID NO: 7; the amino acid sequence of the light chain is shown in SEQ ID NO: 11; the amino acid sequence of HCDR1-3 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 amino acid sequence of the chain is shown in SEQ ID NO: 13.
  • the amino acid sequence of LCDR1 in the antibody or antigen-binding fragment thereof described in the present application may include SEQ ID NO: 15; the amino acid sequence of LCDR2 may include SEQ ID NO: 16; the amino acid sequence of LCDR3 may include SEQ ID NO: 17; and the amino acid sequence of HCDR1 may include SEQ ID NO: 18; the amino acid sequence of HCDR2 may include SEQ ID NO: 19; and the amino acid sequence of HCDR3 may include SEQ ID NO: 20.
  • the antibody or antigen-binding fragment thereof may include antibody SG1202 or an antibody having the same LCDR1-3 and HCDR1-3.
  • the light chain of the antibody or antigen-binding fragment thereof described in the present application may include a light chain variable region, and the amino acid sequence of the light chain variable region may include SEQ ID NO: 21; and 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: 22.
  • the antibody or antigen-binding fragment thereof may include antibody SG1202 or an antibody having the same light chain variable region and heavy chain variable region.
  • the antibody or antigen-binding fragment thereof described in the present application may comprise a light chain and a heavy chain, and the light chain amino acid sequence of the light chain is shown in SEQ ID NO: 25 and the heavy chain amino acid sequence As shown in SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof may include antibody SG1202 or have the same light chain and heavy chain amino acid sequences.
  • the antibody described in this application may be SG1202.
  • the amino acid sequence of LCDR1-3 of antibody SG1202 is shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 21; the amino acid sequence of HCDR1-3 They are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20; the amino acid sequence of VH is shown in SEQ ID NO: 22; the amino acid sequence of light chain is shown in SEQ ID NO: 25; The amino acid sequence of the chain is shown in SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof described in the present application may also include one or more random mutations (e.g., one or more, such as one or more) in the amino acid sequence of the light chain and/or heavy chain of SG1201 and/or SG1202. Several amino acid substitutions).
  • the antibody or its antigen-binding fragment may contain one or more random mutations (e.g., one or more random mutations at one or more positions in the framework region L-FR1-4 of the light chain variable region of SG1201 and/or SG1202).
  • 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 can also be directly (for example, in a frame) 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.
  • the first binding domain, the linker and the second binding domain may be included in sequence.
  • the linker may include the amino acid sequence shown in SEQ ID NO:52.
  • the fusion protein may comprise at least 2, (for example, 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, respectively.
  • the fusion protein may include a first binding domain that specifically binds to PD-L1, and a second binding domain that specifically binds to CD47 protein, wherein the second binding domain may include human SIRP ⁇ variant 1
  • the C-terminus of the mutant, the antibody that specifically binds to PD-L1 or its antigen-binding fragment or variant can be directly or indirectly connected to the N-terminus of the mutant of human SIRP ⁇ variant 1.
  • the second binding domain may comprise at least two human SIRP ⁇ variant 1 mutants, and the N-terminals of the two human SIRP ⁇ variant 1 mutants are respectively connected to an antibody or antigen-binding fragment thereof that specifically binds to PD-L1 or The C-terminal of the variant.
  • the first binding domain of the fusion protein may include SG1201
  • the second binding domain may include two mutant M91 of SIRP ⁇ variant 1
  • the sequence of linker 1 used is as shown in SEQ. ID NO: 52
  • the N-terminals of the two M91s are respectively connected to the C-terminals of the two heavy chains of SG1201 through linker 1.
  • M91 is connected to the C-terminals of the heavy chains of SG1201 to obtain the second
  • the peptide chain, the light chain of SG1201 can be named the first polypeptide chain.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SG12473 are shown in SEQ ID NO: 53 and SEQ ID NO: 11, respectively.
  • the first binding domain of the fusion protein may include SG1202, and the second binding domain may include two SIRP ⁇ variant 1 mutant M91.
  • the sequence of the linker 1 used is as shown in SEQ. ID NO: 52, the N-terminals of the two M91s are respectively connected to the C-terminals of the two heavy chains of SG1202 through linker 1.
  • M91 is connected to the C-terminals of the heavy chains of SG1202 to obtain the second
  • the peptide chain, the light chain of SG1202 can be named the first polypeptide chain.
  • the amino acid sequences of the second polypeptide chain and the first polypeptide chain of SG12474 are shown in SEQ ID NO: 54 and SEQ ID NO: 25, respectively.
  • this application provides one or more isolated nucleic acid molecules that encode the fusion protein or the immunoconjugate.
  • each nucleic acid molecule of the one or more nucleic acid molecules may encode the complete antibody or antigen-binding fragment thereof, or may encode a part thereof (for example, 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 the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • Recombinant DNA and molecular cloning technologies include those developed by Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and TJSilhavy, ML Bennan and LWEnquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM, etc., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987) Those technologies described.
  • the nucleic acids can be prepared from genomic DNA fragments, cDNA, and RNA, and all of these nucleic acids can be directly extracted from cells or produced recombinantly by various amplification methods (including but not limited to PCR and RT-PCR).
  • the direct chemical synthesis of nucleic acids usually involves the sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl groups 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 a phosphotriester, phosphoramidite, and the like. See, for example, Matteuci et al., Tet. Lett. 521:719 (1980); U.S. Patent Nos. 4,500,707 to Caruthers et al.; and U.S. Patent Nos.
  • a vector comprising the isolated polynucleotide of the present application is provided.
  • the vector can be any linear nucleic acid, plasmid, phagemid, cosmid, RNA vector, viral vector, etc.
  • Non-limiting examples of 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, which include the nucleic acid molecule.
  • the vector may contain one or more nucleic acid molecules described in this application. Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • 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 a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • 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 U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part can be fused to a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), and it may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, 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 selectable marker genes, which, after expression, confer one or more phenotypic traits that can be used to select or otherwise identify host cells carrying the vector.
  • selectable marker genes include dihydrofolate reductase and neomycin resistance.
  • the present application provides a cell comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, or the vector.
  • the cell may be a host cell.
  • the cells may include many cell types such as prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 fruit fly cells or Sf9, or fibroblasts, CHO Cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or animal cells of human cells.
  • the vector can be introduced into the host cell stably or transiently by a variety of established techniques.
  • one method involves calcium chloride treatment, in which the carrier is introduced by calcium precipitation.
  • Other salts such as calcium phosphate can also be used in a similar manner.
  • electroporation ie, application of electric current to increase the permeability of cells to nucleic acids
  • transformation methods include microinjection, DEAE dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipoplexes, liposomes and dendrimers can also be used to transfect host cells.
  • heterologous sequence When introducing a heterologous sequence into a host cell, various methods can be implemented to identify the host cell into which the vector has been introduced.
  • An exemplary selection method involves 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 host cells containing heterologous sequences based on the phenotypic traits conferred by the expression of the selectable marker gene contained in the vector.
  • nucleic acid can be prepared from the obtained host cell, and primers specific to the target sequence can be used to amplify the specific target sequence by PCR.
  • 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 is used to detect DNA.
  • nucleic acid probes specific to the target sequence can be used in the hybridization reaction.
  • the expression of a specific gene sequence can be determined by detection of the corresponding mRNA by reverse transcription with PCR, Northern blot hybridization or by immunoassay using an antibody that reacts with the encoded gene product.
  • immunoassays include, but are not limited to, ELISA, radioimmunoassay, and sandwich immunoassay.
  • heterologous sequences of the present application can be confirmed by the enzymatic activity of enzymes (for example, enzymatic markers) encoded by the heterologous sequences.
  • 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 carried out 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 the expression of the fusion protein. For example, it is possible to use an appropriate medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.
  • the method may further include the step of isolating and/or purifying the fusion protein.
  • protein G-Sepharose or Protein A-Sepharose can be used for affinity chromatography, and the fusion protein described in the present application can also be purified and separated by gel electrophoresis and/or high performance liquid chromatography.
  • this application provides an immunoconjugate comprising the fusion protein.
  • the immunoconjugate may be a fusion protein-drug conjugate (ADC), wherein the fusion protein described in the present application is conjugated with one or more therapeutic agents, including but not limited to cytotoxicity Agents, radiotoxic agents (e.g., radioisotopes), and/or immunosuppressive agents (e.g., any agent that kills cells by suppressing immune responses, etc.), etc.
  • the therapeutic agent may be a therapeutic agent capable of treating tumor-related diseases or conditions.
  • the conjugation can be performed through a peptide linker (e.g., a cleavable linker) or other means.
  • the linker can be an acid labile linker, a peptidase sensitive linker, a photolabile linker, and the like.
  • the 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, Nineteenth 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, transdermal administration
  • the medicine is administered via 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 tumor size (or even substantially eliminate the tumor), and/or can reduce and/or stabilize the disease state.
  • the composition described in this application can 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 topical administration of ointment or cream or rectal administration as suppository.
  • the composition may be in unit dosage form suitable for single administration of precise dosages.
  • 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 include 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 disorder or condition (such as a tumor) and/or any complications thereof in a subject suffering from or at risk of development.
  • the specific amount/concentration of the dosage may vary according to the administration method and the needs of the patient, and may be determined based on, for example, the patient's volume, viscosity, and/or weight.
  • a suitable dosage may be about 0.1 mg or 1 mg/kg/day to about 50 mg/kg/day; sometimes, the dosage may be higher. It should be understood that those skilled in the art (for example, a doctor or pharmacist) can conveniently adjust these specific dosages based on the specific patient, formulation, and/or disease condition.
  • treatment or “treatment” or “alleviation” 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 Prevention benefits) methods.
  • therapeutic benefit generally refers to eradicating or reducing the severity of the underlying condition being treated.
  • Treatment benefits for preventive benefits, the composition may be administered to a subject at risk of developing a particular disease, or a subject reporting one or more physiological symptoms of the disease, even if the disease may not have been diagnosed.
  • the 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 medicine , Wherein the drug can be used to treat tumors.
  • the fusion protein, immunoconjugate, nucleic acid molecule, vector, composition or cell described in this application can be used to treat the tumor.
  • the present application provides a method of treating tumors, the method comprising administering to a subject the fusion protein, immunoconjugate, nucleic acid molecule, vector, composition or cell described in the present application.
  • the present application provides a method for blocking the interaction between CD47 protein and SIRP ⁇ , which may include administering (for example, administering to a subject or cell or biological sample in need) Fusion protein or composition.
  • this application provides a method for blocking the interaction between PD-L1 and PD1, and the method may include administration (for example, administration to a subject or cell or biological sample in need) as described in this application
  • administration for example, administration to a subject or cell or biological sample in need
  • the fusion protein or composition may include administration (for example, administration to a subject or cell or biological sample in need) as described in this application.
  • the present application provides a method for inhibiting the growth and/or proliferation of tumors or tumor cells.
  • the method may include contacting the fusion protein or composition described herein with the tumor or tumor cells.
  • the contact can occur in vitro.
  • the present application provides a method that can inhibit 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, and the immunoconjugate Thing, said nucleic acid molecule, said vector, said composition, or said cell.
  • the tumor may include solid tumors and non-solid tumors.
  • the solid tumors and non-solid tumors may include multiple myeloma, leukemia, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, glioma, germ cell tumor, sarcoma, skin Tumor, placental tumor, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, bowel cancer, breast cancer, cervical cancer, ovarian cancer, and testicular cancer , Superior frontal sinus tumor, hypopharyngeal cancer, olfactory blastoma, tongue cancer, gum cancer, ampullary cancer, colon cancer, rectal cancer, kidney cancer, ureteral cancer, bladder cancer, penile cancer, fallopian tube cancer, eyelid cancer, vision Blastoma.
  • the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the composition, or the cell, which can treat tumors or Autoimmune diseases.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, goats, rabbits, mice, rats, or monkeys.
  • the amino acid sequence of the LCDR1-3 of the antibody SG1201 is shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the amino acid sequence of VL is shown in SEQ ID NO: 7; the nucleotides encoding VL The sequence is shown in SEQ ID NO: 9; the amino acid sequence of HCDR1-3 of antibody SG1201 is 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 SG1201 is shown in SEQ ID NO: 11; the nucleotide sequence encoding its light chain is shown in SEQ ID NO: 12.
  • the amino acid sequence of the heavy chain of antibody SG1201 is shown in SEQ ID NO: 13; the nucleotide sequence encoding its heavy chain is shown in SEQ ID NO: 14.
  • the fusion protein SG12473 is composed of a first polypeptide chain and a second polypeptide chain.
  • the first polypeptide chain is the light chain of SG1201. Its amino acid sequence is shown in SEQ ID NO: 11, and the second polypeptide chain has Fc The polypeptide chain obtained by linking the heavy chain of SG1201 with region mutation and M91 through linker 1, the amino acid sequence of which is shown in SEQ ID NO:53.
  • the amino acid sequence of the LCDR1-3 of the antibody SG1202 is shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 21; the nucleotides encoding VL The sequence is shown in SEQ ID NO: 23; the amino acid sequence of HCDR1-3 of antibody SG1202 is shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20; the amino acid sequence of VH is shown in SEQ ID NO: 22; the nucleotide sequence encoding VH is shown in SEQ ID NO: 24.
  • the amino acid sequence of the light chain of antibody SG1202 is shown in SEQ ID NO: 25; the nucleotide sequence encoding its light chain is shown in SEQ ID NO: 26.
  • the amino acid sequence of the heavy chain of antibody SG1202 is shown in SEQ ID NO: 27; the nucleotide sequence encoding its heavy chain is shown in SEQ ID NO: 28.
  • the fusion protein SG12474 is composed of a first polypeptide chain and a second polypeptide chain.
  • the first polypeptide chain is the light chain of SG1202, and its amino acid sequence is shown in SEQ ID NO: 25.
  • the second polypeptide chain has Fc The polypeptide chain obtained by linking the heavy chain of SG1202 with M91 region mutation and M91 through linker 1, its amino acid sequence is shown in SEQ ID NO: 54.
  • the amino acid sequence of IgG1-Fc is shown in SEQ ID NO: 50; the amino acid sequence of Fc with mutation is shown in SEQ ID NO: 51.
  • the mutant M91 (SEQ ID: NO 41) of SIRP ⁇ variant 1 and the IgG1-Fc fusion protein SS002M91 are homodimers, and the amino acid sequence of the monomer is shown in SEQ ID NO: 55.
  • ELISA evaluates the binding activity of the corresponding bifunctional protein and related antigens.
  • Coat PD-L1 human source, PD-L1/B7-H1/CD274Protein (His Tag) purchased from Sino Biological Company
  • PBST 10% fetal bovine serum, Block at 37°C for 1 hour
  • different concentrations of antibody SG1201 and fusion protein SG12473 or, different concentrations of antibody SG1202 and fusion protein SG12474, react at 37°C for 1 hour
  • after washing with PBST add horseradish peroxidase-labeled goat antibody Human IgG secondary antibody (Goat Anti human IgG HRP, Thermo Fisher Scientific), react for 30 minutes at 37°C; wash 5 times with PBST; add 100 ⁇ L TMB (eBioscience) to each well, and place at room temperature (20 ⁇ 5°C) in the dark for 1 to 2 minutes; Then add 100 ⁇ L of 2N H 2 SO 4 stop solution to each well to stop the substrate reaction, read the OD value at 450
  • Figure 2-3 shows that although the fusion protein SG12473 and fusion protein SG12474 have different antibody types for PD-L1, they do not affect the binding ability of fusion protein SG12473 and fusion protein SG12474 to PD-L1.
  • CD47 human source, CD47Protein (His Tag) purchased from Sino Biological Company
  • CD47Protein (His Tag) purchased from Sino Biological Company
  • PBST 10% fetal bovine serum was added and blocked at 37°C for 1 hour
  • Fusion protein SS002M91, fusion protein SG12473 and fusion protein SG12474 were reacted at 37°C for 1 hour
  • horseradish peroxidase-labeled goat anti-human IgG HRP Goat Anti human IgG HRP, Thermo Fisher Scientific
  • TMB eBioscience
  • Figure 4 shows that although the PD-L1 antibody types are different in the fusion protein SG12473 and the fusion protein SG12474, it does not affect the binding ability of the fusion protein SG12473 and the fusion protein SG12474 to CD47.
  • ELISA analyzed the biological activity of the fusion protein SG12473 and the fusion protein SG12474 simultaneously binding to the dual antigens.
  • Figure 5 shows that although the fusion protein SG12473 and the fusion protein SG12474 have different antibody types for PD-L1, it does not affect the ability of the fusion protein SG12473 and the fusion protein SG12474 to simultaneously bind to PD-L1 and CD47.
  • the fusion protein SS002M91 was used as a control to evaluate the biological activity of the fusion protein SG12473 and SG12474 in blocking the CD47/SIRP ⁇ interaction.
  • FIG. 6 shows that the fusion proteins SG12473 and SG12474, like the fusion protein SS002M91, can competitively block the binding of CD47 to its ligand SIRP ⁇ .
  • the IC50 value of SG12473 is 1.26nM
  • the IC50 value of SG12474 is 0.77nM
  • the IC50 value of SS002M91 is 1.16nM.
  • SG1201 and SG1202 were used as controls to evaluate the biological activity of fusion proteins SG12473 and SG12474 in blocking the interaction of PD-1/PD-L1.
  • the PD-L1-Fc was coated with an enzyme-linked plate, 2ug/ml, overnight at 4°C; after washing with PBST, 10% fetal bovine serum was added, and blocked at 37°C for 1 hour; SG1201 was serially diluted with 10% fetal bovine blood.
  • SG12473, SG1202 and SG12474 and add Biotin-Fc-PD1 to the sample to a final concentration of 1ug/ml, and pre-incubate at 37°C for 30 minutes as the primary antibody; after washing the ELISA 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, at room temperature (20 ⁇ 5°C) Keep in the dark for 1-5min; add 100 ⁇ L 2N H 2 SO 4 stop solution to each well to stop the substrate reaction, read the OD value at 450nm by the microplate reader, and analyze the effect of SG1201, SG12473, SG1202 and SG12474 on PD-1/ The blocking effect of PD-
  • the fusion proteins SG12473 and SG12474 can competitively block the binding of PD-1 and PD-L1 like SG1201 and SG1202.
  • SG1201 has an IC50 value of 11.23nM
  • SG12473 has an IC50 value of 13.22nM
  • SG1202 has an IC50 value of 10.89nM
  • SG12474 has an IC50 value of 9.12nM.
  • the human-derived lymphoma KARPAS-299 cell line was subcutaneously xenotransplanted into a female NCG mouse MiXeno animal model to evaluate the effect of the fusion protein SG12473 on tumor activity.
  • mice Select female, 6 to 8 weeks old NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) for experiment. Mice were inoculated with KARPAS-299 cells subcutaneously, and tumor growth was observed regularly. When the tumor grew to an average volume of 35mm 3 , the mice were randomly divided into groups according to tumor size and mouse body weight. On the day of grouping (about Day 4), fresh human PBMC (from a donor) was inoculated via the tail vein to establish a KARPAS-299 humanized mouse model.
  • the experiment was divided into vehicle control group, SG1201 group and SG12473 low, medium and high dose groups, each with 12 mice, and every 6 mice used PBMC from the same source. It is administered by intraperitoneal injection, twice a week, for a total of three weeks. specifically,
  • Group 1A and Group 1B vehicle control group
  • Group 2A and Group 2B SG1201 group, SG1201 5mg/Kg
  • Group 3A and Group 3B SG12473 low-dose group, SG12473 5mg/Kg
  • Group 4A and Group 4B SG12473 middle dose group, SG12473 10mg/Kg
  • Group 5A and Group 5B SG12473 high-dose group, SG12473 20mg/Kg;
  • TGI tumor inhibition rate
  • mice using donor A-derived PBMC the SG12473 low, medium, and high dose groups all showed significant tumor inhibition.
  • the results in Figure 9 show that the TGI of the SG12473 low, medium, and high dose groups were 44.71%, 47.16%, and 96.49% at the completion of the administration, respectively, and the TGIs were 43.71%, 47.92%, and 95.94%, respectively, 4 days after the completion of the administration.
  • the vehicle control group had statistically significant differences (all p values ⁇ 0.01). SG1201 did not show obvious anti-tumor effect at the test dose.
  • mice using donor B-derived PBMC SG12473 low, medium, and high dose groups all showed significant tumor inhibition.
  • the results in Figure 10 show that the TGI of the SG12473 low, medium, and high dose groups were 36.96%, 62.06%, and 98.90% at the completion of the administration, respectively, and the TGIs were 33.44%, 57.57%, and 98.83%, respectively, 4 days after the completion of the administration.
  • the vehicle control group had statistically significant differences (all p values ⁇ 0.01).
  • SG1201 did not show obvious anti-tumor effect at the test dose.

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Abstract

提供一种融合蛋白、与其相关的免疫缀合物、核酸分子、载体、组合物、细胞和制备方法，用于治疗肿瘤和/或自免疫疾病。

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。
程序性死亡蛋白1(programmed death 1,PD-1)抗体是一种免疫疗法。PD-1表达于活化的T细胞，B细胞及髓系细胞，其有两个配体，即程序性死亡分子配体-1(programmed death ligand 1,PD-L1)和PD-L2。PD-1和/或PD-L1抑制剂能够特异性地和肿瘤细胞上的PD-L1结合来达到抗肿瘤作用，然而其临床反应率低，还会带来一些副作用，如引发肺炎、结肠炎、肝炎等。
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PD-L1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PD-L1；2)能够特异性阻断CD47蛋白与SIRPα的相 互作用；3)能够特异性阻断PD-1与PD-L1的相互作用；4)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，所述融合蛋白包含特异性结合PD-L1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:29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、L96、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、L96S/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:30-49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，所述所述PD-L1为人PD-L1。
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包含下组任一项所示的氨基酸序列：SEQ ID NO：4和SEQ ID NO：18。在某些实施方式中，所述HCDR2包含下组任一项所示的氨基酸序列：SEQ ID NO：5和SEQ ID NO：19。在某些实施方式中，所述HCDR3包含下组任一项所示的氨基酸序列：SEQ ID NO：6和SEQ ID NO：20。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包含下组任一项所示的氨基酸序列：SEQ ID NO：8和SEQ ID NO：22。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，所述抗体重链包含下组任一项所示的氨基酸序列：SEQ ID NO：13和SEQ ID NO：27。
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包含下组任一项所示的氨基酸序列：SEQ ID NO：1和SEQ ID NO：15。在某些实施方式中，所述LCDR2包含下组任一项所示的氨基酸序列：SEQ ID NO：2和SEQ ID NO：16。在某些实施方式中，所述LCDR3包含下组任一项所示的氨基酸序列：SEQ ID NO：3和SEQ ID NO：17。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包含下组任一项所示的氨基酸序列：SEQ ID NO：7和SEQ ID NO：21。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，所述抗体轻链包含下组任一项所示的氨基酸序列：SEQ ID NO：11和SEQ ID NO：25。
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：52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。
在某些实施方式中，所述肿瘤包括实体瘤和非实体瘤。
在某些实施方式中，所述实体瘤和非实体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、上额窦瘤、下咽癌、嗅母细胞瘤、舌癌、牙龈癌、壶腹癌、结肠癌、直肠癌、肾癌、输尿管癌、膀胱癌、阴茎癌、输卵管癌、眼睑癌、视母细胞瘤。
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。
另一方面，本申请提供了一种阻断PD-L1蛋白与PD-1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。
另一方面，本申请提供了一种预防或治疗受试者中的肿瘤的方法，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，其预防或治疗受试者中的肿瘤。
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2-3显示的是本申请所述融合蛋白与PD-L1的结合能力。
图4显示的是本申请所述融合蛋白与CD47的结合能力。
图5显示的是本申请所述融合蛋白与PD-L1和CD47同时结合的能力。
图6显示的是本申请所述融合蛋白竞争性阻断CD47与其配体SIRPα的结合。
图7-8显示的是本申请所述融合蛋白竞争性阻断PD-1与PD-L1的结合。
图9-10显示的是利用本申请所述的融合蛋白在小鼠人淋巴瘤肿瘤模型中抑制肿瘤体积的增长。
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:29所示。
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，PD-L1)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.,1988 Science 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.,1989Proc Natl Acad Sci USA 86:10029-10033；Co,et al.,1991Proc 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
在本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，特异性结合PD-L1蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，特异性结合PD-L1蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。 对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“PD-L1”通常是指程序性死亡蛋白-1(programmed death-1,PD-1)蛋白的配体，其又可称为CD274、B7-H或B7H1。PD-1通过与特异性配体(PD-L)相互作用负调节T细胞抗原受体信号传导。所述PD-L1可以为多种肿瘤的预后指标。在本申请中，所述PD-L1可以为人PD-L1。所述人PD-L1在GenBank的Gene ID为29126。
在本申请中，术语“PD-1”通常是指突触核蛋白家族的成员，其又可称为NACP、PARK1或PARK4。在本申请中，所述PD1可以为人PD1。所述人PD1在GenBank的Gene ID为6622。
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“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PD-L1；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:29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PD-L1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。
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、L96、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:29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:29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:29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、L96S/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:29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:29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:29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:30-49中任一项所示的氨基酸序列。
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:41所示的氨基酸序列。
特异性结合PD-L1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。
本申请所述的抗体或其抗原结合片段，可通过与PD-L1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PD-L1阳性的肿瘤。如，所述PD-L1阳性的肿瘤可以选自以下组：胃癌、乳腺癌、宫颈癌、肺癌、头颈部肿瘤、黑素瘤、胶质瘤、淋巴上皮瘤、食管癌或结直肠癌。在本申请中，所述抗体、其抗原结合片段可以杀伤胃癌、乳腺癌、宫颈癌、肺癌、头颈部肿瘤、黑素瘤、胶质瘤、淋巴上皮瘤、食管癌或结直肠癌细胞或抑制胃癌、乳腺癌、宫颈癌、肺癌、头颈部肿瘤、黑素瘤、胶质瘤、淋巴上皮瘤、食管癌或结直肠癌细胞生长。
本申请所述的PD-L1蛋白可为人PD-L1蛋白或其功能片段。例如，所述的PD-L1蛋白可以不为小鼠PD-L1蛋白，或者可以不为大鼠PD-L1蛋白。在某些实施方式中，本申请所述的抗体、其抗原结合片段基本上不结合小鼠PD-L1蛋白或大鼠PD-L1蛋白。
本申请所述的抗体、其抗原结合片段，可以与参比抗体竞争结合所述PD-L1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可包含下组任一项所示的氨基酸序列：SEQ ID NO：1和SEQ ID NO：15；所述LCDR2可包含下组任一项所示的氨基酸序列：SEQ ID NO：2和SEQ ID NO：16；所述LCDR3可包含下组任一项所示的氨基酸序列：SEQ ID NO：3和SEQ ID NO：17。所述参比抗体的重链可变区可包含HCDR1-3，所述HCDR1可包含下组任一项所示的氨基酸序列：SEQ ID NO：4和SEQ ID NO：18；所述HCDR2可包含下组任一项所示的氨基酸序列：SEQ ID NO：5和SEQ ID NO：19；所述HCDR3可包含下组任一项所示的氨基酸序列：SEQ ID NO：6和SEQ ID NO：20。
例如，所述参比抗体的轻链可变区的氨基酸序列可以包含下组任一项所示的氨基酸序列：SEQ ID NO：7和SEQ ID NO：21，且所述参比抗体的重链可变区的氨基酸序列可以包含下组任一项所示的氨基酸序列：SEQ ID NO：8和SEQ ID NO：22。又例如，所述参比抗体的轻链可包含下组任一项所示的氨基酸序列：SEQ ID NO：11和SEQ ID NO：25；且所述参比抗体的重链可包含下组任一项所示的氨基酸序列：SEQ ID NO：13和SEQ ID NO：27。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:25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:27所示的氨基酸序列。
本申请所述的抗体、其抗原结合片段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。又例如，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:15。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:16。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:17。
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的氨基酸序列可以为：SEQ ID NO:11。又例如，所述轻链可变区VL的氨基酸序列可以为：SEQ ID NO:21。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:25。
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。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。又例如，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:18。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 19。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:20。
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如下的氨基酸序列：SEQ ID NO:13。又例如，所述重链可变区VH可包含如下的氨基酸序列：SEQ ID NO:22。在某些实施方式中，所述抗体重链可包含如下的氨基酸序列：SEQ ID NO:27。
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:25；并且其重链的氨基酸序列包括SEQ ID NO:27。
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SG1201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SG1201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SG1201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SG1201。抗体SG1201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；轻链的氨基酸序列如SEQ ID NO:11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；重链的氨基酸序列如SEQ ID NO:13所示。
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:15；LCDR2的氨基酸序列可包括SEQ ID NO:16；LCDR3的氨基酸序列可包括SEQ ID NO:17；且HCDR1的氨基酸序列可包括SEQ ID NO:18；HCDR2的氨基酸序列可包括SEQ ID NO:19；HCDR3的氨基酸序列可包括SEQ ID NO:20。例如，该抗体或其抗原结合片段可包括抗体SG1202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:21；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:22。例如，该抗体或其抗原结合片段可包括抗体SG1202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:25所示且所述重链氨基酸序列如SEQ ID NO:27所示。例如，该抗体或其抗原结合片段可包括抗体SG1202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SG1202。抗体SG1202的LCDR1-3的氨基酸序列分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示；VL的氨基酸序列如SEQ ID NO:21所示；HCDR1-3的氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和 SEQ ID NO:20所示；VH的氨基酸序列如SEQ ID NO:22所示；轻链的氨基酸序列如SEQ ID NO:25所示；重链的氨基酸序列如SEQ ID NO:27所示。
本申请所述的抗体或其抗原结合片段，还可以在SG1201和/或SG1202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、其抗原结合片段可在SG1201和/或SG1202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SG1201和/或SG1202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)。
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:52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PD-L1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可以包含人SIRPα变体1的突变体，特异性结合PD-L1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PD-L1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(SG12473)的第一结合域可以包含SG1201，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:52所示，2个M91的N端分别通过连接子1连接于SG1201的2条重链的C端，在所述融合蛋白 中，M91连接SG1201重链的C端可以得到第二多肽链，SG1201的轻链可以命名为第一多肽链。SG12473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:53和SEQ ID NO:11所示。
例如，如图1所示，所述融合蛋白(SG12474)的第一结合域可以包含SG1202，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:52所示，2个M91的N端分别通过连接子1连接于SG1202的2条重链的C端，在所述融合蛋白中，M91连接SG1202重链的C端可以得到第二多肽链，SG1202的轻链可以命名为第一多肽链。SG12474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:54和SEQ ID NO:25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。
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。
另一方面，本申请提供了治疗肿瘤的方法，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，本申请提供了一种阻断PD-L1与PD1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。
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。
在某些实施方式中，所述肿瘤可以包括实体瘤和非实体瘤。
在某些实施方式中，所述实体瘤和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、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、上额窦瘤、下咽癌、嗅母细胞瘤、舌癌、牙龈癌、壶腹癌、结肠癌、直肠癌、肾癌、输尿管癌、膀胱癌、阴茎癌、输卵管癌、眼睑癌、视母细胞瘤。
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。
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。
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构建融合蛋白
参照如图1所示的融合蛋白结构，以罗氏PD-L1抗体Atezolizumab(SG1201)、SIRPα变体1的突变体M91(SEQ ID:NO 41)为例，从N端到C端，选用选择连接子1(SEQ ID:NO 52)，依次连接SG1201、连接子和2个M91，其中2个M91的N端分别和SG1201的重链的C端连接，得到融合蛋白SG12473。
抗体SG1201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SG1201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SG1201的轻链的氨基酸序列如SEQ ID NO:11所示；编码其轻链的核苷酸序列如SEQ ID NO:12所示。抗体SG1201的重链的氨基酸序列如SEQ ID NO:13所示；编码其重链的核苷酸序列如SEQ ID NO:14所示。
所述融合蛋白SG12473由第一多肽链和第二多肽链构成，第一多肽链即SG1201的轻链，其氨基酸序列如SEQ ID NO:11所示，第二多肽链即具备Fc区突变的SG1201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:53所示。
参照如图1所示的融合蛋白结构，以阿斯利康PD-L1抗体Durvalumab(SG1202)、SIRPα变体1的突变体M91(SEQ ID:NO 41)为例，从N端到C端，选用选择连接子1(SEQ ID:NO 52)，依次连接SG1202、连接子和2个M91，其中2个M91的N端分别和SG1202的重链的C端连接，得到融合蛋白SG12474。
抗体SG1202的LCDR1-3的氨基酸序列分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示；VL的氨基酸序列如SEQ ID NO:21所示；编码VL的核苷酸序列如SEQ ID NO:23所示；抗体SG1202的HCDR1-3的氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示；VH的氨基酸序列如SEQ ID NO:22所示；编码VH的核苷酸序列如SEQ ID NO:24所示。抗体SG1202的轻链的氨基酸序列如SEQ ID NO:25所示；编码其轻链的核苷酸序列如SEQ ID NO:26所示。抗体SG1202的重链的氨基酸序列如SEQ ID NO:27所示；编码其重链的核苷酸序列如SEQ ID NO:28所示。
所述融合蛋白SG12474由第一多肽链和第二多肽链构成，第一多肽链即SG1202的轻链，其氨基酸序列如SEQ ID NO:25所示，第二多肽链即具备Fc区突变的SG1202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:54所示。
其中，IgG1-Fc的氨基酸序列如SEQ ID NO:50所示；具备突变的Fc的氨基酸序列如SEQ  ID NO:51所示。
SIRPα变体1的突变体M91(SEQ ID:NO 41)与IgG1-Fc融合蛋白SS002M91为同二聚体，其单体的氨基酸序列如SEQ ID NO:55所示。
实施例2与双抗原结合的活性检测
(1)以抗不同抗原的人源化抗体为对照，ELISA评价相应双功能蛋白和相关抗原的结合活性。
将PD-L1(人源，购买自Sino Biological公司的PD-L1/B7-H1/CD274Protein(His Tag))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SG1201、融合蛋白SG12473；或者，不同浓度的抗体SG1202、融合蛋白SG12474，37℃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，分析双功能蛋白与相关靶抗原结合能力。
结果如图2-3所示。图2-3显示融合蛋白SG12473和融合蛋白SG12474中虽然PD-L1的抗体种类不同，但并不影响融合蛋白SG12473和融合蛋白SG12474与PD-L1的结合能力。
(2)以CD47受体突变体M91为对照，ELISA评价相应双功能蛋白和CD47的结合活性。
将CD47(人源，购买自Sino Biological公司的CD47Protein(His Tag))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、融合蛋白SG12473和融合蛋白SG12474，37℃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SG12473和融合蛋白SG12474与CD47结合能力。
结果如图4所示。图4显示融合蛋白SG12473和融合蛋白SG12474中虽然PD-L1的抗体种类不同，但并不影响融合蛋白SG12473和融合蛋白SG12474与CD47的结合力。
实施例3同时与双抗原结合的活性检测
以抗不同抗原的人源化抗体为对照，ELISA分析融合蛋白SG12473和融合蛋白SG12474同时结合双抗原的生物学活性。
将PD-L1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；分别加入不同浓度的抗体SG1201、融合蛋白SG12473、抗体SG1202、融合蛋白SG12474，37℃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SG12473和融合蛋白SG12474与PD-L1和CD47同时结合的能力。
结果如图5所示，图5显示融合蛋白SG12473和融合蛋白SG12474中虽然PD-L1的抗体种类不同，但并不影响融合蛋白SG12473和融合蛋白SG12474与PD-L1和CD47同时结合的能力。
实施例4阻断CD47/SIRPα相互作用的活性分析
以融合蛋白SS002M91为对照，评价融合蛋白SG12473、SG12474阻断CD47/SIRPα相互作用的生物学活性。
将SIRPα-His包被酶联板，1ug/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、SG12473、SG12474，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、SG12473、SG12474对CD47/SIRPα的阻断作用。
结果如图6所示，图6显示融合蛋白SG12473、SG12474与融合蛋白SS002M91一样可以竞争性阻断CD47与其配体SIRPα的结合。其中SG12473的IC50值为1.26nM，SG12474的IC50值为0.77nM，SS002M91的IC50值为1.16nM。
实施例5阻断PD-1/PD-L1相互作用的活性分析
以SG1201、SG1202为对照，评价融合蛋白SG12473、SG12474阻断PD-1/PD-L1相互作用的生物学活性。
将PD-L1-Fc包被酶联板，2ug/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SG1201、SG12473、SG1202和SG12474，并在样品中加入Biotin-Fc-PD1至终浓度1ug/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SG1201、SG12473、SG1202和SG12474对PD-1/PD-L1的阻断作用。
从图7-8中可以看出，融合蛋白SG12473、SG12474与SG1201、SG1202一样可以竞争性阻断PD-1与PD-L1的结合。其中SG1201的IC50值为11.23nM，SG12473的IC50值为13.22nM，SG1202的IC50值为10.89nM，SG12474的IC50值为9.12nM。
实施例6融合蛋白体内抑制肿瘤活性
人源淋巴瘤KARPAS-299细胞株皮下异种移植雌性NCG小鼠MiXeno动物模型，评价融合蛋白SG12473抑制肿瘤活性的效果。
选择雌性，6～8周龄的NCG小鼠(购自江苏集萃药康生物技术有限公司)进行试验。小鼠于皮下接种KARPAS-299细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积35mm 3时根据肿瘤大小和小鼠体重随机分组给药。分组当天(约Day4)经尾静脉接种新鲜人PBMC(来源于捐献者)，建立KARPAS-299人源化小鼠模型。
试验分为溶媒对照组、SG1201组以及SG12473低、中、高剂量组，每组12只，每6只小鼠使用同一来源的PBMC。腹腔注射给药，每周给药两次，共给药三周。具体地，
组1A和组1B：溶媒对照组
组2A和组2B：SG1201组，SG1201 5mg/Kg
组3A和组3B：SG12473低剂量组，SG12473 5mg/Kg
组4A和组4B：SG12473中剂量组，SG12473 10mg/Kg
组5A和组5B：SG12473高剂量组，SG12473 20mg/Kg；
根据相对肿瘤抑制率(TGI)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
结果显示，使用捐献者A来源的PBMC的小鼠中，SG12473低、中、高剂量组均表现出显著的抑瘤作用。图9的结果显示，SG12473低、中、高剂量组在给药完成时TGI分别为44.71％、47.16％和96.49％，在给药完成4天后TGI分别为43.71％、47.92％和95.94％，相对溶媒对照组统计学上均有显著性差异(p值均<0.01)。SG1201在测试剂量下没有表现出明显的抑瘤作用。
使用捐献者B来源的PBMC的小鼠中，SG12473低、中、高剂量组均表现出显著的抑瘤 作用。图10的结果显示，SG12473低、中、高剂量组在给药完成时TGI分别为36.96％、62.06％和98.90％，在给药完成4天后TGI分别为33.44％、57.57％和98.83％，相对溶媒对照组统计学上均有显著性差异(p值均<0.01)。SG1201在测试剂量下没有表现出明显的抑瘤作用。
各治疗组均无动物死亡，没有表现明显的药物毒性，治疗期间耐受良好。
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 (41)

  1. 融合蛋白，其包含：
    特异性结合PD-L1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:29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、L96、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)I6、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)I6、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、L96S/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:30-49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。
  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，dAb，分离的互补决定区CDR，Fv和scFv。
  10. 根据权利要求1-9中任一项所述的融合蛋白，其中所述PD-L1为人PD-L1。
  11. 根据权利要求7-10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包含下组任一项所示的氨基酸序列：SEQ ID NO：4和SEQ ID NO：18。
  12. 根据权利要求11所述的融合蛋白，其中所述HCDR2包含下组任一项所示的氨基酸序列：SEQ ID NO：5和SEQ ID NO：19。
  13. 根据权利要求11-12中任一项所述的融合蛋白，其中所述HCDR3包含下组任一项所示的氨基酸序列：SEQ ID NO：6和SEQ ID NO：20。
  14. 根据权利要求11-13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可变区VH，且所述重链可变区VH包含下组任一项所示的氨基酸序列：SEQ ID NO：8和SEQ ID NO：22。
  15. 根据权利要求11-14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。
  16. 根据权利要求15所述的融合蛋白，其中所述IgG选自下组：IgG1和IgG4。
  17. 根据权利要求11-16中任一项所述的融合蛋白，其中所述抗体重链包含下组任一项所示的氨基酸序列：SEQ ID NO：13和SEQ ID NO：27。
  18. 根据权利要求6-17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包含下组任一项所示的氨基酸序列：SEQ ID NO：1和SEQ ID NO：15。
  19. 根据权利要求18所述的融合蛋白，其中所述LCDR2包含下组任一项所示的氨基酸序列：SEQ ID NO：2和SEQ ID NO：16。
  20. 根据权利要求18-19中任一项所述的融合蛋白，其中所述LCDR3包含下组任一项所示的氨基酸序列：SEQ ID NO：3和SEQ ID NO：17。
  21. 根据权利要求18-20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可变区VL，且所述轻链可变区VL包含下组中任一项所示的氨基酸序列：SEQ ID NO:7和SEQ ID NO：21。
  22. 根据权利要求18-21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18-22中任一项所述的融合蛋白，其中所述抗体轻链包含下组任一项所 示的氨基酸序列：SEQ ID NO：11和SEQ ID NO：25。
  24. 根据权利要求1-23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。
  25. 根据权利要求1-24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。
  26. 根据权利要求25所述的融合蛋白，其中所述连接子包含如SEQ ID NO:52所示的氨基酸序列。
  27. 根据权利要求1-26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。
  28. 根据权利要求27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。
  29. 免疫缀合物，其包含根据权利要求1-28中任一项所述的融合蛋白。
  30. 一个或多个分离的核酸分子，其编码根据权利要求1-28中任一项所述的融合蛋白或者根据权利要求29所述的免疫缀合物。
  31. 一个或多个载体，其包含权利要求30所述的核酸分子。
  32. 组合物，其包含根据权利要求1-28中任一项所述的融合蛋白，根据权利要求30所述的免疫缀合物，或根据权利要求30所述的核酸分子，以及任选地药学上可接受的赋形剂。
  33. 细胞，其包含权利要求1-28中任一项所述的融合蛋白，权利要求29所述的免疫缀合物，权利要求30所述的核酸分子，或权利要求31所述的载体。
  34. 制备根据权利要求1-28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33所述的细胞。
  35. 权利要求1-28中任一项所述的融合蛋白，权利要求29所述的免疫缀合物，权利要求30所述的核酸分子，权利要求31所述的载体，权利要求32所述的组合物，或权利要求33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。
  36. 根据权利要求35所述的用途，其中所述肿瘤包括实体瘤和非实体瘤。
  37. 根据权利要求36所述的用途，其中所述实体瘤和非实体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、上额窦瘤、下咽癌、嗅母细胞瘤、舌癌、牙龈癌、壶腹癌、结肠癌、直肠癌、肾癌、输尿管癌、膀胱癌、阴茎癌、输卵管癌、眼睑癌、视母细胞瘤。
  38. 权利要求1-28中任一项所述的融合蛋白，权利要求29所述的免疫缀合物，权利要求 30所述的核酸分子，权利要求31所述的载体，权利要求32所述的组合物，或权利要求33所述的细胞，其治疗肿瘤。
  39. 阻断PD-L1蛋白与PD-1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1-28中任一项所述的融合蛋白，权利要求29所述的免疫缀合物，权利要求30所述的核酸分子，权利要求31所述的载体，权利要求32所述的组合物，或权利要求33所述的细胞。
  40. 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1-28中任一项所述的融合蛋白，权利要求29所述的免疫缀合物，权利要求30所述的核酸分子，权利要求31所述的载体，权利要求32所述的组合物，或权利要求33所述的细胞。
  41. 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权利要求1-28中任一项所述的融合蛋白，权利要求29所述的免疫缀合物，权利要求30所述的核酸分子，权利要求31所述的载体，权利要求32所述的组合物，或权利要求33所述的细胞。
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