Fusion proteins that bind to CD47 protein and uses thereof

Abstract

The present invention is -8 The present invention provides a fusion protein capable of binding to CD47 protein with a KD value of M or lower, and uses thereof. The fusion protein can specifically block the interaction between CD47 protein and SIRPα, does not cause a blood coagulation reaction, and further suppresses the growth and / or proliferation of tumors or tumor cells. [Selection diagram] None

Description

[Technical Field]

[0001] Technical area The present invention relates to a fusion protein that binds to CD47 protein and its use, which can specifically block the interaction between CD47 protein and SIRPα, does not induce blood coagulation, and inhibits the growth and / or proliferation of tumors or tumor cells.

[0002] Background technology The CD47 protein is a member of the immunoglobulin superfamily and a transmembrane glycoprotein expressed on the surface of many cells, including red blood cells. CD47 ligands include integrins, thrombospondin-1, and signal-regulating proteins (SIRPs). CD47 influences multiple biological functions, including cell migration, T cell and dendritic cell activation, and axon growth. Additionally, CD47 interacts with SIRPα to inhibit phagocytosis by phagocytes, protecting normal cells, such as blood cells, from engulfment by phagocytes. Research has shown that in addition to CD47 expression on normal tissue cells, many tumor cells overexpress CD47 and bind to SIRPα on the surface of phagocytes, thereby preventing tumor cell engulfment by phagocytes, potentially providing a tumor evasion mechanism for immune surveillance. Blocking the interaction between CD47 protein and SIRPα can suppress tumor growth (Theocharides APA, et al., 2012).

[0003] However, existing reagents used to block the interaction between CD47 and SIRPα have limited recognition activity, insufficient affinity for CD47, and limited tumor suppression. Meanwhile, existing antibody-based drugs targeting CD47 have side effects such as anemia and thrombocytopenia (Bai Yinpeng et al., Chin J Clin Oncol., 2017 Vol. 44, No. 7). Novel therapeutic approaches with minimal side effects that can effectively block the interaction between CD47 and SIRPα are urgently needed.

[0004] Invention Contents The present invention provides a fusion protein that binds to CD47 protein and uses thereof. The fusion protein can specifically bind to CD47 protein. The fusion protein described in the present invention has at least one of the following properties: 1) specifically binds to CD47 protein with high affinity, 2) specifically blocks the interaction between CD47 protein and SIRPα, 3) does not cause blood coagulation, 4) suppresses the growth and / or proliferation of tumors or tumor cells, 5) inhibits apoptotic signals induced by CD47 / SIRPα interaction, and / or 6) is safe for subjects and has no harmful side effects. The present invention also provides methods for preparing and using the fusion protein.

[0005] On the other hand, the present invention provides a method for producing a antibody capable of specifically binding to CD47 protein and having a concentration of 1) 1×10 -8 M or lower K D The present invention provides a fusion protein having at least one of the following characteristics: 1) binding to CD47 protein at a specific level; 2) specifically blocking the interaction between CD47 protein and SIRPα; 3) not causing a blood coagulation reaction; and 4) suppressing the growth and / or proliferation of tumors or tumor cells.

[0006] In one embodiment, the CD47 protein is a human CD47 protein.

[0007] In one embodiment, the CD47 protein is a CD47 protein expressed on the cell surface.

[0008] In certain embodiments, the tumor or tumor cells are CD47 positive.

[0009] In one embodiment, the tumor is selected from the group of CD47-positive hematological tumors and / or CD47-positive solid tumors.

[0010] In one embodiment, the fusion protein contains a human SIRPα domain capable of specifically binding to the CD47 protein and an immunoglobulin Fc region, wherein the human SIRPα domain and the immunoglobulin Fc region are linked directly or indirectly.

[0011] In certain embodiments, the human SIRPα domain comprises the extracellular domain of human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein.

[0012] In certain embodiments, the human SIRPα domain comprises the IgV domain of human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein.

[0013] In certain embodiments, the human SIRPα domain comprises the human SIRPα variant 1 domain, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein.

[0014] In certain embodiments, the human SIRPα domain comprises the IgV domain of human SIRPα variant 1, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein.

[0015] In one embodiment, the human SIRPα domain comprises amino acid residues 33 to 149 of human SIRPα variant 1, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein.

[0016] In one embodiment, the human SIRPα domain described in the present invention comprises an amino acid sequence set forth in any of SEQ ID NOs: 1-20, 62-65, and an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto.

[0017] In one embodiment, a fusion protein according to the invention comprises an amino acid sequence set forth in any of SEQ ID NOs: 21-61, and an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto.

[0018] In one embodiment, the human SIRPα domain, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein comprises a substitution, deletion, or addition of one or several amino acid residues.

[0019] In one embodiment, the mutant contains an amino acid substitution at one or more residues selected from the group of I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109 and V132.

[0020] In one embodiment, the mutant has one or more amino acid substitutions selected from the group of R22C, I29L, I61L / V / F, V63I, E77I / N / Q / K / H / M / R / N / V / L, Q82S / R / G / N, K83R, E84K / H / D / R / G, V93L / A, D95H / R / E, D96S / T, K98R, N100G / K / D / E, R107N / S, G109R / H and V132L / R / I / S.

[0021] In one embodiment, the human SIRPα domain comprises an amino acid sequence represented by any of SEQ ID NOs: 1-20, 62-65.

[0022] In one embodiment, the immunoglobulin Fc region comprises an IgG Fc region.

[0023] In one embodiment, the IgG is human IgG. In one embodiment, the IgG is selected from the group of IgG1 and / or IgG4.

[0024] In one embodiment, the human SIRPα domain is located at the N-terminus of the immunoglobulin Fc region.

[0025] In one embodiment, the human SIRPα domain and the immunoglobulin Fc region are connected by a linker.

[0026] In one embodiment, the immunoglobulin Fc region comprises an amino acid sequence represented by any of SEQ ID NOs: 67-68.

[0027] In one embodiment, the fusion protein comprises an amino acid sequence represented by any of SEQ ID NOs: 21-61.

[0028] On the other hand, the present invention provides a nucleic acid molecule encoding a fusion protein according to the present invention.

[0029] On the other hand, the present invention provides a vector comprising a nucleic acid molecule according to the present invention.

[0030] On the other hand, the present invention provides a host cell comprising a nucleic acid molecule according to the invention or a vector according to the invention.

[0031] On the other hand, the present invention provides a method for preparing a fusion protein according to the present invention, which comprises culturing a host cell according to the present invention under conditions that allow expression of said fusion protein.

[0032] On the other hand, the present invention provides a composition comprising a fusion protein, a nucleic acid molecule, a vector and / or a host cell according to the present invention, and optionally a pharmaceutically acceptable adjuvant.

[0033] Meanwhile, the present invention provides the use of the fusion protein, nucleic acid molecule, vector, host cell and / or composition described in the present invention in the preparation of a medicament and / or kit for the prevention or treatment of tumors or autoimmune diseases. In one embodiment, the tumor is selected from the group of CD47-positive hematologic tumors or CD47-positive solid tumors. In one embodiment, the autoimmune disease is selected from the group of Crohn's disease, allergic asthma and rheumatoid arthritis.

[0034] On the other hand, the present invention provides a method for blocking the interaction of CD47 protein and SIRPα, comprising administering a fusion protein or composition according to the present invention.

[0035] On the other hand, the present invention provides a method for inhibiting the growth and / or proliferation of a tumor or tumor cells, comprising contacting the tumor or tumor cells with a fusion protein or composition described in the present invention. In one embodiment, the contacting is performed in vitro.

[0036] Meanwhile, the present invention provides a method for preventing or treating a tumor or an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of a fusion protein or composition described in the present invention. In one embodiment, the tumor is selected from the group consisting of CD47-positive hematologic tumors or CD47-positive solid tumors. In one embodiment, the autoimmune disease is selected from the group consisting of Crohn's disease, allergic asthma, and rheumatoid arthritis.

[0037] Those skilled in the art will readily appreciate other aspects and advantages of the present disclosure from the following detailed description. The following detailed description depicts and describes only exemplary embodiments of the present disclosure. Those skilled in the art will appreciate that the details of the present disclosure may alter the specific embodiments disclosed without departing from the spirit and scope of the present invention. Accordingly, the drawings and description of the present invention are merely illustrative and are not intended to limit the present invention. [Brief explanation of the drawings]

[0038] Attached diagram explanation Specific features of the present invention are set forth in the appended claims. The features and advantages of the present invention will be more clearly understood by reference to the following detailed description of exemplary embodiments and drawings, which are generally described as follows:

[0039] FIG. 1 shows a schematic diagram of the physical structure of the vector pTM. FIG. 2 shows a schematic diagram of the method for measuring the interaction between SIRPα truncated domains and their mutants and CD47. FIG. 3 shows the results of a flow cytometry enrichment screen of SIRPα truncated domain mutants. FIG. 4 shows a sequence alignment of SIRPα truncated domains and their variants. FIG. 5 shows the results of the recognition of CD47 protein by the fusion protein according to the present invention. 6A-6B show the specificity with which the fusion protein according to the present invention recognizes human CD47 protein. FIG. 7 shows the mode of recognition of human CD47 protein and other proteins by the fusion protein according to the present invention. FIG. 8 shows that the fusion protein according to the present invention competitively blocks the binding of CD47 protein to its ligand SIRPα with TTI-621. 9A to 9C show the results of the fusion protein according to the present invention recognizing the CD47 protein on the surface of Raji cells, Jurkat cells, and A549 cells. 10A to 10C show the results of the recognition of CD47 protein on the surface of Raji cells, Jurkat cells, and A549 cells by the fusion protein described in the present invention and TTI-621. FIG. 11 shows the results of the anticoagulant reaction between the fusion protein according to the present invention and Hu5F9-G4. 12A-12B show the inhibition of anti-tumor activity by the fusion protein according to the present invention. 13A-13B show a comparison of the effects of a fusion protein according to the present invention and TTI-621 on red blood cells and platelets. FIG. 14 shows the results of the recognition of CD47 protein by the fusion protein according to the present invention. FIG. 15 shows that the fusion proteins described in the present invention competitively block the binding of CD47 protein to its ligand SIRPα. FIG. 16 shows the results of the anticoagulant reaction between the fusion protein according to the present invention and Hu5F9-G4. FIG. 17 shows that the fusion protein according to the invention effectively blocks CD47-Fc-induced Jurkat-CSR cell apoptosis. FIG. 18A shows the effect of the fusion protein according to the invention on red blood cell content in mouse peripheral blood. FIG. 18B shows the effect of the fusion protein according to the invention on platelet content in peripheral blood of mice. FIG. 19 shows the tumor growth suppression role in mice in vivo by the fusion protein according to the present invention. FIG. 20 shows the effect of the fusion protein according to the invention on the body weight of mice.

[0040] Specific embodiment Hereinafter, embodiments of the present invention will be described with reference to certain specific examples, but those skilled in the art will be able to easily understand other benefits and effects of the present invention from the contents disclosed in this specification.

[0041] fusion proteins On the other hand, the present invention provides a method for producing a medicament capable of specifically binding to CD47 protein, comprising the steps of: -8 M or lower K D value, e.g., 9 x 10 -9 No higher than M, 8 x 10 -9 No higher than M, 7 x 10 -9 No higher than M, 6.2 x 10 -9 No higher than M, 6 x 10 -9 No higher than M, 5 x 10 -9 Not higher than M, 4.8 x 10 -9 No higher than M, 4.5 x 10 -9 No higher than M, 2 x 10 -9 No higher than M, 1.5 x 10 -9 No higher than m or 1 x 10 -10 K not higher or lower than M D The present invention provides a fusion protein that binds to the CD47 protein at a specific binding level.

[0042] The fusion proteins described in the present invention may activate tumor cell phagocytosis by phagocytes or suppress certain cell apoptosis signals by specifically blocking the interaction between CD47 protein and SIRPα. The fusion proteins described in the present invention do not induce blood clotting reactions. For example, they are tested on a blood clotting plate by placing the fusion protein and a red blood cell solution on the plate and allowing the red blood cells to sink to the bottom of the holes without spreading flat like a mesh. The fusion proteins can also inhibit the growth and / or proliferation of tumors or tumor cells, for example, by reducing tumor area or tumor volume or improving the survival rate of tumor-affected subjects. Furthermore, the fusion proteins can be safely administered to subjects without negatively affecting the subject's weight and / or mortality. The fusion proteins described in the present invention are easy to prepare and obtain, regardless of the specific immunoglobulin Fc region.

[0043] In the present invention, the term fusion protein generally refers to a composite polypeptide, i.e., a single contiguous amino acid sequence consisting of two (or more) polypeptides. Fusion proteins are generally obtained by artificial preparation using recombinant nucleic acid methods or chemical synthesis methods.

[0044] In the present invention, the term CD47 protein, also referred to as integrin-associated protein (IAP), belongs to the immunoglobulin superfamily. The CD47 protein can bind to membrane integrins, thrombospondin-1 (TSP-1), or signal-regulatory protein alpha (SIRPα). The CD47 protein can be expressed on the cell membrane surface. The CD47 protein may be a supramolecular complex consisting of a specific integrin protein, a G protein, and cholesterol. In the present invention, the CD47 protein may be human CD47 protein with GenBank database accession number CEJ95640.1. In the present invention, the CD47 protein may contain the amino acid sequence represented by SEQ ID NO:66.

[0045] In the present invention, the term "CD47-positive" generally refers to the characteristics of the CD47 protein, its fragments, or its mutants in which one or more amino acids are substituted, expressed in vivo or on the cell surface. CD47-positive cells may be cells that overexpress CD47. Typically, the CD47-positive cells may be used as disease indicators. For example, in the case of disease, the CD47 protein density on the surface of the CD47-positive cells exceeds the CD47 protein density of such cells under normal conditions. In one embodiment, the tumor or tumor cells may be CD47-positive. For example, the tumor may be selected from the group of CD47-positive hematological tumors and / or CD47-positive solid tumors.

[0046] In the present invention, K D The term K may be used interchangeably with KD and refers to the equilibrium dissociation constant of a given antibody-antigen interaction, usually in units of M (mol / L). D may be calculated from the concentrations of substance AB and the substances A and B that dissociated it. D=c(A)*c(B) / c(AB). According to this formula, K D The larger the value, the more dissociation occurs, and the weaker the affinity between substances A and B becomes. D The smaller the value, the less dissociation there is and the stronger the affinity between substances A and B.

[0047] In the present invention, the term SIRPα generally refers to a regulatory membrane glycoprotein from the SIRP family. SIRPα can recognize CD47 protein as a ligand for the CD47 protein. SIRPα is a transmembrane protein with three immunoglobulin superfamily-like domains in the extracellular region, the N-terminal region of which mediates binding to CD47. SIRPα is mainly expressed on the surface of phagocytes, dendritic cells, and neurons. The cytoplasmic region of SIRPα is highly conserved among rats, mice, and humans. Although SIRPα exhibits polymorphism, this does not affect its recognition and binding to CD47 protein.

[0048] In the present invention, the term "human SIRPα domain" generally includes human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein. In the present invention, the human SIRPα domain may include the extracellular domain of human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein. The human SIRPα domain may include the IgV domain of human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein. The human SIRPα domain may include the domain of human SIRPα variant 1, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein. The human SIRPα domain may include amino acid residues 33 to 149 of human SIRPα variant 1, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein. In humans, SIRPα proteins mainly exist in two forms: one is (human SIRPα variant 1 or V1 type) whose amino acid sequence has GenBank accession number NP_542970.1 (the amino acid sequence is represented by SEQ ID NO: 62, in which amino acid residues 31 to 504 constitute the mature SIRPα domain), and the other is (variant 2 or V2 type) which contains 13 amino acids different from variant 1 or V1 type and whose amino acid sequence has GenBank accession number CAA71403.1.

[0049] In the present invention, the human SIRPα domain may comprise the extracellular domain of human SIRPα, a fragment thereof, or a mutant thereof in which one or more amino acids are substituted therein.

[0050] In the present invention, the term "extracellular domain" generally refers to a functional domain of a protein located outside the cell membrane. In one embodiment, the extracellular domain refers to the extracellular domain of a human SIRPα domain, a fragment thereof, or a mutant thereof in which one or more amino acids are substituted therein. For example, the extracellular domain of the human SIRPα domain may contain three immunoglobulin superfamily (IgSF) domains and multiple glycosylation sites. The extracellular domain of the human SIRPα domain can achieve its signaling function by specifically binding to a ligand (e.g., CD47 protein). The extracellular domain of the human SIRPα domain is phosphorylated by activation with multiple mitogens, such as serum, insulin, growth factors, EGF, PDGF, and neurotrophic factors.

[0051] In the present invention, the human SIRPα domain may comprise the IgV domain of human SIRPα, a fragment thereof, or a mutant thereof in which one or more amino acids have been substituted therein. In the present invention, the human SIRPα domain may comprise human SIRPα variant 1, a fragment thereof, or a mutant thereof in which one or more amino acids have been substituted therein. For example, the human SIRPα domain may comprise amino acid residues 33 to 149 of human SIRPα variant 1, a fragment thereof, or a mutant thereof in which one or more amino acids have been substituted therein.

[0052] In the present invention, the term "IgV domain" generally refers to an Ig-like domain similar to an antibody variable domain. Immunoglobulin domains may be divided into four types: IgV, IgC1, IgC2, and IgI. IgV domains may exist in different protein families, including immunoglobulin light and heavy chains, T cell receptors, etc. Human SIRPα exhibits high polymorphism in the IgV domain. For example, the IgV domain of human SIRPα variant 1 can mediate the binding of the human SIRPα domain to human CD47 protein (Seiffert, M. et al. (2001) Blood 97, 2741-9; Vernon-ffilson, EF et al. (2000) Eur J Immunol 30, 2130-7).

[0053] For example, the human SIRPα variant 1, a fragment thereof, or a mutant thereof having one or more amino acid substitutions therein may contain the amino acid sequence set forth in SEQ ID NO: 62. For example, the human SIRPα domain may include the IgV domain of human SIRPα variant 1, a fragment thereof, or a mutant thereof having one or more amino acid substitutions therein. For example, the IgV domain of human SIRPα variant 1, a fragment thereof, or a mutant thereof having one or more amino acid substitutions therein may contain the amino acid sequence set forth in SEQ ID NO: 65 (i.e., residues 38 to 145 of the amino acid sequence set forth in SEQ ID NO: 62). Also, for example, the human SIRPα domain may include a truncated domain of human SIRPα variant 1, a fragment thereof, or a mutant thereof having one or more amino acid substitutions therein. The truncated domain of human SIRPα variant 1, a fragment thereof, or a mutant thereof in which one or more amino acids are substituted therein may contain the amino acid sequence represented by SEQ ID NO: 63 (i.e., residues 33 to 149 of the amino acid sequence represented by SEQ ID NO: 62).

[0054] The human SIRPα domain according to the present invention may comprise an amino acid sequence represented by any one selected from SEQ ID NOs: 1-20, 62-65.

[0055] In the present invention, the term "mutant" generally refers to a mutated protein, polypeptide, or amino acid sequence. The mutation may indicate a difference from the wild-type. For example, the mutation may result in a structural change in the amino acid sequence relative to the wild-type. For example, the wild-type may be a representative phenotype of the lack of the structural change. For example, the mutant may be a mutant obtained by mutating a human SIRPα domain and a fragment thereof (including the IgV domain of human SIRPα, a fragment thereof, the domain of human SIRPα variant 1, a fragment thereof, or amino acid residues 33 to 149 of human SIRPα variant 1, a fragment thereof) as the wild-type.

[0056] In the present invention, the mutant may contain amino acid substitutions at one or more residues selected from the group consisting of I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109, and V132. In the amino acid substitutions, the amino acid residue position may be determined by a residue number based on the amino acid sequence represented by SEQ ID NO: 62. Herein, "residue Xn" refers to residue X corresponding to the nth residue in the amino acid sequence represented by SEQ ID NO: 62, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "residue I61" refers to amino acid residue I corresponding to the 61st residue in the amino acid sequence represented by SEQ ID NO: 62.

[0057] In the present invention, "amino acid substitution Xn" refers to an amino acid substitution at residue X corresponding to the nth residue in the amino acid sequence represented by SEQ ID NO:62, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "amino acid substitution I61" refers to an amino acid substitution at amino acid residue I corresponding to the 61st residue in the amino acid sequence represented by SEQ ID NO:62.

[0058] In the present invention, the expression "corresponding" to an amino acid residue in one amino acid sequence in another amino acid sequence generally refers to an amino acid residue correspondence obtained by amino acid sequence alignment under optimized conditions. The sequence alignment is performed using methods well known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms necessary to achieve optimal alignment of the full-length sequences being compared.

[0059] Amino acid substitutions according to the present invention may be non-conservative substitutions, which change amino acid residues in a target protein or polypeptide in a non-conservative manner, for example, changing an amino acid residue having one type of side chain size or one type of property (e.g., hydrophilicity) to an amino acid residue having a different side chain size or different property (e.g., hydrophobicity).

[0060] The amino acid substitutions may be conservative substitutions. Such conservative substitutions may conservatively alter amino acid residues in the target protein or polypeptide, for example, by changing an amino acid residue having a certain side chain size or certain properties (e.g., hydrophilicity) to an amino acid residue having the same or similar side chain size or properties (e.g., hydrophilicity). Such conservative substitutions usually do not significantly affect the structure or function of the produced protein. In the present invention, the amino acid sequence variants of the fusion protein, its fragments, or mutants thereof in which one or more amino acids are substituted therein may contain conservative amino acid substitutions that do not appreciably alter the protein structure or its function (e.g., the ability to block binding of CD47 to its ligand).

[0061] For example, in each of the following groups, mutual substitutions between amino acids within each group are considered conservative substitutions in the present invention: The group of amino acids containing nonpolar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. A group of amino acids containing uncharged polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. A group of amino acids containing negatively charged polar side chains: aspartic acid and glutamic acid. Positively charged basic amino acids: lysine, arginine and histadine. Amino acids with a phenyl group: phenylalanine, tryptophan, and tyrosine.

[0062] In certain embodiments, the mutant may comprise one or more amino acid substitutions selected from the group consisting of I61L / V / F, V63I, E77I / N / Q / K / H / M / R / N / V / L, Q82S / R / G / N, K83R, E84K / H / D / R / G, V93L / A, D95H / R / E, D96S / T, K98R, N100G / K / D / E, R107N / S, G109R / H and V132L / R / I / S.

[0063] In the present invention, the amino acid substitution "XnY / Z" refers to a substitution of amino acid residue Y or Z for residue X corresponding to the nth residue in the amino acid sequence represented by SEQ ID NO: 62, where n is a positive integer, X, Y, and Z are each independently an abbreviation for any amino acid residue, and X is different from Y or Z. For example, the amino acid substitution "I61L / V / F" refers to a substitution of amino acid residue L, V, or F for residue I corresponding to the 61st residue in the amino acid sequence represented by SEQ ID NO: 62.

[0064] For example, the fusion protein according to the present invention may be (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, D95H, L96S, G109H and V132S, (11) I61F, D95H, L96S, K98R, G109H and V132S, (12) I61L, E77Q, E84D, V93A, R107N and V132I, (13) E77K, L96S, N100K, G109H and V132L, (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D and V132I, (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D and V132L, (16) I61V, E77N, Q82S, K83R, E84H and V93A, (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R and N100E, (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D and G109R, (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R and N100G, and (20) I61L, V63I, E77N, Q82G and E84G, It may have amino acid substitutions selected from:

[0065] In the present invention, based on the truncated domain of human SIRPα variant 1 (e.g., the amino acid sequence represented by SEQ ID NO: 63, i.e., residues 33 to 149 in the amino acid sequence represented by SEQ ID NO: 62), SIRPα domain variants having the following amino acid substitution groups (1) to (20) are designated M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111, M122, M126, M130, M135, and M145, respectively. These mutants may contain, for example, any of the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 20, respectively.

[0066] In the present invention, a fusion protein comprising a truncated domain of human SIRPα variant 1 (e.g., the amino acid sequence represented by SEQ ID NO: 63, i.e., residues 33 to 149 in the amino acid sequence represented by SEQ ID NO: 62) and human IgG1 Fc (e.g., the amino acid sequence represented by SEQ ID NO: 67) may be designated, for example, as SS002, containing the amino acid sequence represented by SEQ ID NO: 61.

[0067] In the present invention, the term immunoglobulin Fc region generally refers to the base region of the Y-shaped antibody structure, also referred to as the fragment crystallizable region (Fc region). In IgG, IgA, and IgD antibody isoforms, the Fc region may be composed of two identical protein fragments from the second and third constant domains of the two heavy chains of the antibody. The Fc region of IgM and IgE may contain three heavy chain constant domains per polypeptide chain. The IgG Fc region has highly conserved N-glycosylation sites. In some embodiments, the immunoglobulin Fc region may comprise an IgG Fc region. In some embodiments, the immunoglobulin Fc region may comprise the CH2 and CH3 regions of the heavy chain constant region. In some embodiments, the immunoglobulin Fc region may comprise a hinge region. For example, the immunoglobulin Fc region may comprise an amino acid sequence selected from any of SEQ ID NOs: 67-68.

[0068] In the present invention, the term IgG generally refers to immunoglobulin G. IgG is one of the human immunoglobulins. Based on differences in the antigenicity of the γ chain in the IgG molecule, human IgG has four subtypes: IgG1, IgG2, IgG3, and IgG4. In the present invention, the term IgG1 generally refers to the subtype that has high affinity for Fc receptors and accounts for the largest proportion of IgG. For example, the IgG may be human IgG. Also, for example, the IgG may be selected from the group of IgG1 and / or IgG4.

[0069] In the present invention, the fusion protein may comprise a human SIRPα domain capable of specifically binding to the CD47 protein and an immunoglobulin Fc region, wherein the human SIRPα domain is directly or indirectly linked to the immunoglobulin Fc region. For example, the human SIRPα domain may be located at the N-terminus of the immunoglobulin Fc region. For example, the C-terminus of the human SIRPα domain may be directly or indirectly linked to the N-terminus of the immunoglobulin Fc region. For example, the human SIRPα domain may be linked to the immunoglobulin Fc region via a linker. In the present invention, the linker may be a peptide linker.

[0070] In the present invention, based on the above SS002, the fusion proteins described in the present invention containing any one of the amino acid substitution groups (1) to (20) above may be named SS002M1, SS002M5, SS002M12, SS002M35, SS002M37, SS002M41, SS002M57, SS002M67, SS002M81, SS002M82, SS002M84, SS002M91, SS002M99, SS002M102, SS002M111, SS002M122, SS002M126, SS002M130, SS002M135, and SS002M145, respectively. These fusion proteins may, for example, contain the amino acid sequences represented by SEQ ID NO: 21-SEQ ID NO: 40, respectively.

[0071] In the present invention, a truncated domain of human SIRPα variant 1 (the amino acid sequence represented by SEQ ID NO: 63, i.e., including residues 33 to 149 in the amino acid sequence represented by SEQ ID NO: 62) is prepared by substituting any one of the amino acid substitution groups (1) to (20) above, and a truncated domain of human IgG4 Fc (e.g., SEQ ID NO: 68) may be successively named SS002M1G4, SS002M5G4, SS002M12G4, SS002M35G4, SS002M37G4, SS002M41G4, SS002M57G4, SS002M67G4, SS002M81G4, SS002M82G4, SS002M84G4, SS002M91G4, SS002M99G4, SS002M102G4, SS002M111G4, SS002M122G4, SS002M126G4, SS002M130G4, SS002M135G4 and SS002M145G4. These fusion proteins may contain, for example, the amino acid sequences set forth in any of SEQ ID NO: 41-SEQ ID NO: 60, in order.

[0072] In one embodiment, the fusion protein according to the present invention may comprise an amino acid sequence represented by any of SEQ ID NO: 21-SEQ ID NO: 61.

[0073] It should be understood that the proteins, polypeptides and / or amino acid sequences according to the present invention include variants or homologues that have at least the same or similar function as the proteins or polypeptides.

[0074] In the present invention, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of the protein and / or polypeptide (e.g., a human SIRPα domain, a fragment thereof, or a mutant thereof in which one or more amino acids have been substituted therein, or the fusion protein). For example, the functional variant may include a protein or polypeptide having an amino acid modification by substitution, deletion, and / or insertion of at least one, e.g., 1 to 30, 1 to 20, or 1 to 10, or e.g., 1, 2, 3, 4, or 5 amino acids. The functional variant can essentially retain the biological properties of the protein or polypeptide before the alteration (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., the ability to specifically bind to CD47 protein) of the protein or polypeptide before the alteration.

[0075] In the present invention, the homologue may be a protein or polypeptide having at least about 85% (e.g., 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) sequence homology with the amino acid sequence of the protein and / or polypeptide (e.g., the human SIRPα domain, a fragment thereof, or a mutant thereof in which one or more amino acids are substituted therein, or the fusion protein).

[0076] In the present invention, the term "homology" generally refers to the similarity, similarity, or correlation between two or more sequences. "Percent sequence identity" refers to the number of positions in the two sequences to be aligned that contain identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) across a comparison window. The number of matching positions is calculated by dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percent sequence identity. Alignment to determine percent sequence identity can be achieved by various means well known in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, and Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including the total sequence coverage being compared and any algorithms needed to achieve maximum alignment in the sequence region of interest. Homology can also be measured using the FASTA and BLAST algorithms. The FASTA algorithm is described in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Search for Protein Similarity," Science, 227:1435-1441, 1989. The BLAST algorithm is described in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "Basic Local Alignment Search Tools," Journal of Molecular Biology, 215:403-410, 1990.

[0077] Nucleic acid molecules, vectors, and host cells On the other hand, the present invention provides one or more nucleic acid molecules capable of encoding the fusion proteins according to the present invention.

[0078] In one embodiment, the nucleic acid molecule can completely encode the fusion protein described in the present invention. For example, the fusion protein can be obtained using a single nucleic acid molecule. In one embodiment, the nucleic acid molecule can encode a portion of the fusion protein described in the present invention. For example, the fusion protein can be obtained using two or more different nucleic acid molecules. For example, the nucleic acid molecule can encode the human SIRPα domain in the fusion protein described in the present invention (e.g., the extracellular domain of the human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein; the IgV domain of the human SIRPα, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein; or the domain of the human SIRPα variant 1, a fragment thereof, or a mutant thereof with one or more amino acid substitutions therein). Alternatively, for example, the nucleic acid molecule can encode the immunoglobulin Fc region in the fusion protein.

[0079] On the one hand, the present invention provides one or more vectors comprising one or more nucleic acid molecules according to the present invention, and on the other hand, the present invention provides a cell (e.g., a host cell) comprising a nucleic acid molecule according to the present invention or a vector according to the present invention.

[0080] In the present invention, the term "nucleic acid molecule" generally refers to nucleotides, deoxyribonucleotides, or ribonucleotides, or their analogs, of any length, in isolated form, isolated from their natural environment or artificially synthesized. The nucleic acid molecule described in the present invention may be isolated. For example, it may be produced or synthesized by (i) in vitro amplification, e.g., polymerase chain reaction (PCR), (ii) clonal recombination, (iii) purification, e.g., enzymatic digestion and gel electrophoretic fractionation, or (iv) synthesis, e.g., chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology. In the present invention, nucleic acids encoding the above-described antibodies or antigen-binding fragments thereof can be prepared by various techniques well known in the art, including, but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. Specific guidance can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0081] In the present invention, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host and transporting an inserted nucleic acid molecule into and / or between host cells. Such vectors include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcribing and / or translating DNA or RNA. The vectors also include vectors with multiple functions. The vectors may be polynucleotides that can be transcribed and translated into polypeptides when introduced into suitable host cells. The vectors typically produce desired expression products by culturing suitable host cells containing the vector. In the present invention, the vectors may contain one or more of the nucleic acid molecules. For example, the vector may contain all the nucleic acid molecules necessary to encode the fusion protein. In this case, only one vector is required to obtain the fusion protein described in the present invention. In one embodiment, the vector may contain a single nucleic acid molecule encoding a portion of the fusion protein, such as a nucleic acid molecule encoding the human SIRPα domain of the fusion protein described in the present invention, or a nucleic acid molecule encoding the immunoglobulin Fc region of the fusion protein. In this case, two or more different vectors are required to obtain the fusion protein according to the present invention.

[0082] The vector may further include other genes, such as a marker gene that allows the vector to be selected in a suitable host cell under suitable conditions. The vector may further include expression regulatory elements that allow the coding region to be properly expressed in an appropriate host. Such regulatory elements include, for example, promoters, ribosome binding sites, enhancers, and other regulatory elements that control gene transcription and mRNA translation, and are well known to those skilled in the art. In some embodiments, the expression regulatory sequence is regulatable. The specific structure of the expression regulatory sequence varies depending on the function of the organism or cell type, but typically includes a 5' non-transcribed sequence and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, such as a TATA box, capping sequence, and CAAT sequence. For example, the 5' non-transcribed expression regulatory sequence may include a promoter region that includes a promoter sequence for controlling the transcription of an operably linked nucleic acid. In the present invention, the vector may be a pTM vector.

[0083] In the present invention, the terms host cell, cell, and host are used interchangeably and generally refer to a single cell, cell line, or cell culture that can contain or already contains a plasmid or vector comprising a nucleic acid molecule described in the present invention, or that can express a fusion protein, fragment, or mutant thereof described in the present invention. Such a cell may include progeny of a single host cell. The progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell due to natural, unintended, or intentional mutations, but they may express an antibody or antigen-binding fragment thereof described in the present invention. The host cell described above can be obtained by in vitro transfecting cells with a vector described in the present invention. The host cell described above may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell, COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). In the present invention, the host cell may be a CHO cell.

[0084] Compositions, methods of preparation and uses On the other hand, the present invention can provide a method for preparing the fusion protein, which comprises culturing a host cell under conditions that allow the fusion protein to be expressed.

[0085] On the other hand, the present invention can provide a composition comprising the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned vector and / or the above-mentioned host cell, and optionally a pharmaceutically acceptable adjuvant.

[0086] In the present invention, the term pharmaceutically acceptable adjuvant may include buffering agents, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or non-ionic surfactants, etc.

[0087] The pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or non-ionic surfactants, etc.

[0088] In the present invention, the pharmaceutical compositions are prepared and manipulated together with pharmaceutically acceptable vectors or diluents and any other known adjuvants and excipients according to conventional means in the art, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0089] In the present invention, the composition can be prepared for administration by oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, intravaginal administration, transdermal administration or subcutaneous storage.

[0090] In the present invention, the compositions may be used to inhibit tumor growth, for example, the compositions of the present invention may inhibit or slow the development or progression of a disease (e.g., a tumor or an autoimmune disease) (e.g., by reducing tumor size and thus essentially eliminating the tumor) and / or reduce and / or stabilize the disease state.

[0091] A pharmaceutical composition according to the present invention may comprise a therapeutically effective amount of the fusion protein, the therapeutically effective amount being a dose for preventing and / or treating (at least partially treating) a disease (e.g., a tumor or an autoimmune disease) and / or any complications thereof in a subject at risk of developing the disease.

[0092] On the other hand, the present invention provides the use of the fusion protein, nucleic acid molecule, vector, host cell and / or composition according to the present invention in the preparation of a medicament and / or kit for the prevention or treatment of tumors or autoimmune diseases.

[0093] In the present invention, the term "tumor" generally refers to a new product formed in a living body by localized tissue cell hyperplasia. Because such new products often consist of space-occupying blocks, they are also called neoplasms. Depending on the cellular characteristics of the new product and the degree of harmfulness to the living body, tumors are divided into two types: benign tumors and malignant tumors, and malignant tumors are collectively referred to as cancer. The tumors described in the present invention may be selected from the group consisting of CD47-positive hematologic tumors and / or CD47-positive solid tumors.

[0094] In the present invention, the term "CD47-positive hematologic tumor" generally refers to hematologic tumors that overexpress CD47, including various leukemias, lymphomas, and myelomas. Leukemia is a blood cancer in which excessive production of white blood cells, which do not normally contribute to infection prevention, overwhelms other components of the blood, such as platelets and red blood cells. Leukemia can be classified as acute or chronic. Leukemia can include, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myeloproliferative disorders / neoplasms (MPDS), and myelodysplastic syndromes. Lymphoma can include Hodgkin's lymphoma, indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). The myeloma may be referred to as multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, light chain myeloma, or Bence-Jones myeloma.

[0095] In the present invention, the term "CD47-positive solid tumor" refers to a solid tumor or tumor mass that overexpresses CD47 and can be detected by clinical examination, such as X-ray, CT scan, B-mode ultrasound image, or palpation. It is mainly classified into carcinoma and sarcoma. For example, the CD47-positive solid tumor can include Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, astrocytoma, glioblastoma multiforme, and renal cell carcinoma.

[0096] In the present invention, the autoimmune diseases may include Crohn's disease, allergic asthma and rheumatoid arthritis.

[0097] In the present invention, the term Crohn's disease generally refers to a type of inflammatory bowel disease of unknown cause that can occur anywhere in the gastrointestinal tract. Crohn's disease and chronic nonspecific ulcerative colitis are collectively referred to as inflammatory bowel disease (IBD).

[0098] In the present invention, the term allergic asthma generally refers to chronic airway inflammation involving multiple cell types, particularly mast cells, eosinophilic granulocytes and T lymphocytes.

[0099] In the present invention, the term rheumatoid arthritis generally refers to one of the chronic systemic autoimmune diseases primarily characterized by arthropathy.

[0100] On the other hand, the fusion proteins, nucleic acid molecules, vectors, host cells and / or compositions according to the present invention are used to prevent or treat said tumors or said autoimmune diseases.

[0101] On the other hand, the present invention provides a method for preventing or treating a tumor or an autoimmune disease, comprising administering to a subject a fusion protein, a nucleic acid molecule, a vector, a host cell and / or a composition according to the present invention.

[0102] On the other hand, the present invention provides a method for blocking the interaction of CD47 protein and SIRPα, comprising administering (e.g., to a subject or a cell or biological sample in need thereof) a fusion protein or composition described in the present invention.

[0103] On the other hand, the present invention provides a method for inhibiting the growth and / or proliferation of a tumor or tumor cells, comprising contacting the tumor or tumor cells with a fusion protein or composition described in the present invention, for example, in vitro.

[0104] In the present invention, the term subject generally refers to any human or non-human animal. The term non-human animal may include all vertebrates, such as mammals and non-mammals, such as non-human primates, goats, sheep, dogs, cows, birds, amphibians, reptiles, etc.

[0105] In the present invention, the term "about" generally refers to a range of 0.5%-10% above or below a given numerical value, for example, 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%, or 10% above or below the given numerical value.

[0106] In the present invention, the term "comprise" generally refers to including, comprising, containing or having, and in some cases also means being or consisting of.

[0107] The following examples are intended to illustrate the features, methods and systems of the present invention without relying on any theory, and are not intended to limit the scope of the present invention.

[0108] Example Example 1: Selection of mutants A truncated domain of human SIRPα variant 1 (NP_542970.1), whose amino acid sequence is represented by SEQ ID NO: 63 (i.e., residues 33 to 149 in SEQ ID NO: 62), was used to construct the interaction structure of the truncated domain with human CD47 (CEJ95640.1) using Discovery Studio (Chuangteng Technology) software. The sites involved in the interaction between the two proteins and the interaction model were theoretically analyzed. The following amino acid sites in the truncated domain were identified as directly or indirectly involved in the interaction with CD47: I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109, and V132 (the positions of these amino acid substitutions were numbered according to the amino acid sequence represented by SEQ ID NO: 62). Random mutations were introduced into these active sites to construct a mutant library. The mutant library was then cloned into the vector pTM, which contains a signal peptide and a transmembrane domain sequence (e.g., as shown in Figure 1) and enables expression of genes cloned into the vector on the cell surface.

[0109] The constructed mutant library expression vector was transfected into CHO cells (ATCC) to express the mutant library on the cell surface. CD47 protein (Shenzhou Biological Technology Dexian Liability Company) was then labeled with FITC to obtain CD47-FITC. The enrichment of mutants capable of binding to CD47-FITC was then detected using flow cytometry based on the binding strength between CD47-FITC and the truncated domain mutants on the CHO cell surface. The specific detection principle can be seen in Figure 2, which shows the binding of the truncated domain and mutants to the fluorescent molecule-containing CD47 protein as a function of the level of fluorescent molecules.

[0110] After four rounds of enrichment detection, cells with strong CD47-FITC binding (e.g., as shown in Figure 3) were collected. The mRNA was extracted and transcribed to obtain cDNA, and the genes of the truncated domain mutants were sequenced (e.g., as shown in Figure 4). Sequencing results showed the presence of different combinations of mutations at the following sites: I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109, and V132.

[0111] The results showed that by introducing a combination of different mutation sites into residues I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109 and / or V132, mutants of the human SIRPα variant 1 truncated domain that can specifically recognize CD47 can be obtained.

[0112] Further analysis identified the following amino acid types after possible mutations at the above mutation sites: I61L / V / F, V63I, E77I / N / Q / K / H / M / R / N / V / L, Q82S / R / G / N, K83R, E84K / H / D / R / G, V93L / A, D95H / R / E, D96S / T, K98R, N100G / K / D / E, R107N / S, G109R / H, and V132L / R / I / S.

[0113] The mutants containing these mutations are designated M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111, M122, M126, M130, M135, and M145, respectively, and each contains, in order, an amino acid sequence represented by, for example, any of SEQ ID NOs: 1-20, and each has, for example, a sequence similar to that represented by, for example, SEQ ID NO: 63, based on the amino acid sequence represented by, for example, SEQ ID NO: 63. (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, D95H, L96S, G109H and V132S, (11) I61F, D95H, L96S, K98R, G109H and V132S, (12) I61L, E77Q, E84D, V93A, R107N and V132I, (13) E77K, L96S, N100K, G109H and V132L, (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D and V132I, (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D and V132L, (16) I61V, E77N, Q82S, K83R, E84H and V93A, (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R and N100E, (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D and G109R, (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R and N100G, and (20) I61L, V63I, E77N, Q82G and E84G The amino acid mutation combinations are sequentially

[0114] Example 2 Measurement of fusion protein binding activity The truncated domain of human SIRPα variant 1 in Example 1 (also referred to as wild-type SIRPα truncated domain) and the mutants of the human SIRPα domain obtained in Example 1 (i.e., M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111, M122, M126, M130, M135 and M145) were each purified using human IgG1-Fc (the amino acid sequences of which are shown in, for example, SEQ ID NO: 1). The truncated domains of SIRPα mutant 1 were fused with and expressed with the corresponding truncated domains of SIRPα mutant 1-human Fc fusion proteins (abbreviated as fusion proteins), and these fusion proteins were designated SS002, SS002M1, SS002M5, SS002M12, SS002M35, SS002M37, SS002M41, SS002M57, SS002M67, SS002M81, SS002M82, SS002M84, SS002M91, SS002M99, SS002M102, SS002M111, SS002M122, SS002M126, SS002M130, SS002M135, and SS002M145, respectively. These fusion proteins contain the amino acid sequences represented by any of SEQ ID NOs: 61, 21-40, respectively.

[0115] [Table 1]

[0116] As examples, the fusion proteins SS002, SS002M12, SS002M5, SS002M82, SS002M84, SS002M91, SS002M102, and SS002M130 were selected for biological activity analysis.

[0117] The affinity of each fusion protein, such as SS002, SS002M5, SS002M12, SS002M82, SS002M84, SS002M91, SS002M102, and SS002M130, to the CD47 molecule was measured using ELISA.

[0118] ELISA strips were coated with 1 μg / ml of the target antigen CD47-His and incubated overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added and blocked at 37°C for 1 hour. SS002, SS002M5, SS002M82, SS002M84, SS002M91, SS002M102, and SS002M130 were then added and incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-conjugated goat anti-human IgG secondary antibody (Goat Anti-human IgG HRP, Thermo Fisher Scientific) was added and incubated at room temperature for 30 minutes. The plates were then washed five times with PBST and the remaining liquid was absorbed with blotting paper. 100 ml of TMB (eBioscience) was added to each well, and the plate was left in the dark at room temperature (20 ± 5°C) for 1 to 5 minutes. 100 ml of 2N H2SO4 was added to each well to stop the substrate reaction. OD values ​​at 450 nm were read using a microplate reader, and the affinity of each fusion protein for the CD47 molecule was analyzed (e.g., as shown in Figure 5).

[0119] The results in Figure 5 show that each of the fusion proteins, such as SS002, SS002M5, SS002M12, SS002M82, SS002M84, SS002M91, SS002M102, and SS002M130, was able to effectively recognize the CD47 molecule.

[0120] Example 3 Affinity Analysis For example, the affinity of each fusion protein, such as SS002, SS002M5, SS002M12, SS002M82, SS002M84, SS002M91, SS002M102, and SS002M130, to the CD47 molecule was measured using the Biacore method, and the results are shown in Table 2 below.

[0121] [Table 2]

[0122] The results in Table 2 show that each of the fusion proteins, such as SS002, SS002M5, SS002M12, SS002M82, SS002M84, SS002M91, SS002M102, and SS002M130, was able to recognize the CD47 molecule with high affinity.

[0123] Example 4 Fusion protein species recognition specificity The specific recognition activity was analyzed using the fusion proteins SS002 and SS002M91 as examples.

[0124] To perform species analysis of the fusion proteins, ELISA strips were coated with 1 mg / mL human CD47 and mouse CD47 (Shenzhou Biological Technology Dexian Liability Company) and left overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added and blocked at 37°C for 1 hour. SS002 and SS002M91 were then added and incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-conjugated goat anti-human IgG secondary antibody (Goat Anti-human IgG HRP, Thermo Fisher Scientific) was added and incubated at room temperature for 30 minutes. The plates were then washed five times with PBST, and residual droplets were absorbed with blotting paper. Then, 100 ml of TMB (eBioscience) was added to each well, and the plates were left at room temperature (20 ± 5°C) in the dark for 1 to 5 minutes. 100 ml of 2N H2SO4 was added to each well to stop the substrate reaction. The OD values ​​at 450 nm were read using a microplate reader, and the binding ability of the fusion proteins to heterologous CD47 was analyzed (the experimental results for the fusion proteins SS002 and SS002M91 are shown in Figures 6A and 6B, respectively).

[0125] The results in Figures 6A and 6B show that both SS002 and SS002M91 were able to specifically recognize the human CD47 molecule, but did not recognize the mouse CD47 molecule.

[0126] Example 5 Specific Recognition of Fusion Proteins for Target Antigens Taking the fusion proteins SS002 and SS002M91 as an example, 1 mg / ml of SS002, SS002M91, milk (Beijing Bomeide Biotechnology Co., Ltd), BSA (BOVOGEN), CD19 (Shenzhou Biological Technology Dexian Liability Company), TROP2 (Shenzhou Biological Technology Dexian Liability Company), CD47 (Beijing Maigeboer Biological Technology Co., Ltd.), CD38 (Shenzhou Biological Technology Dexian Liability Company), Gas6 (R&D), and other proteins, as well as AXL (ACRO ELISA strips were coated with eBiosystems (Biosystems) and incubated overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added and blocked at 37°C for 1 hour. SS002 or SS002M91 was added and incubated at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-conjugated goat anti-human IgG secondary antibody (Goat Anti-human IgG HRP, Thermo Fisher Scientific) was added and incubated at room temperature for 30 minutes. The plates were then washed five times with PBST. Any remaining droplets were absorbed with blotting paper. 100 ml of TMB (eBioscience) was added to each well and incubated at room temperature (20±5°C) for 1-5 minutes in the dark. 100 ml of 2N H2SO4 was added to each well to stop the substrate reaction. The OD at 450 nm was measured using a microplate reader to analyze the binding affinity of the fusion proteins to each of the above proteins (see, for example, Figure 7).

[0127] The results in FIG. 7 show that the fusion proteins SS002 and SS002M91 only recognized the human CD47 molecule and did not cross-react with any other proteins.

[0128] Example 6 Specific Blockade of CD47 / SIRPα Interaction by Fusion Proteins SS002M91 was used as an example to analyze its activity of specifically blocking the CD47 / SIRPα interaction, and TTI-621 (see CN105073780A), which expresses the American congener, was used as a positive control.

[0129] ELISA strips were coated with 1 μg / ml SIRPα-His and left overnight at 4°C. After washing with PBST, 10% fetal bovine serum was added and blocked for 1 hour at 37°C. SS002M91 and TTI-621 were diluted with 10% fetal bovine serum and then pre-incubated for 30 minutes at 37°C with Biotin-Fc-CD47 as the primary antibody, with a final concentration of 2 μg / ml. After washing the ELISA strips with PBST, the primary antibody was added and incubated for 1 hour at 37°C. After washing five times with PBST, horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiaxuan Biotechnology Co., Ltd.) was added and incubated at 37°C for 30 minutes. After washing five times with PBST, 100 μL of TMB (eBioscience) was added per well and the plate was left at room temperature (20±5°C) in the dark for 1 to 5 minutes. 100 μL of 2N H2SO4 was added per well to stop the substrate reaction. The OD value at 450 nm was read using a microplate reader to analyze the blocking effect of CD47 / SIRPα by the SIRPα fusion protein (as shown in Figure 8).

[0130] The results in Figure 8 indicate that both SS002M91 and TTI-621 could competitively block the binding of CD47 to its ligand SIRPα, but the blocking activity of the fusion protein SS002M91 was significantly higher than that of TTI-621. The IC50 values ​​for SS002M91 and TTI-621 were 5.47 μg / mL and 493.5 μg / mL, respectively.

[0131] Example 7 Specific recognition of CD47 molecules on the surface of tumor cells by fusion proteins Using the fusion proteins SS002 and SS002M91 as examples, we analyzed their recognition activity for CD47 molecules on the surface of tumor cells.

[0132] The specific recognition activity of SS002 and SS002M91 on the surface of Raji cells, Jurkat cells, and A549 cells was measured using a flow cytometer (BD Calibur). The cells were harvested from the logarithmic growth phase and grown at a cell density of 5 × 10 6 The solution was adjusted to cells / mL and pre-chilled on ice for 10 minutes. SIRPα fusion proteins SS002 and SS002M91 were diluted to different concentrations in pre-chilled saline containing 2% FBS. 100 μL of cells were added with the same volume of the diluted SIRPα fusion protein and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with pre-chilled saline containing 2% FBS. The cells were resuspended in 100 μL of diluted PE-conjugated goat anti-human IgG-Fc secondary antibody (PE-Goat anti-human IgG Fc Secondary Antibody, eBioscience) and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed twice with pre-chilled saline containing 2% FBS. The cells were resuspended in 400 μL of 1% polyformaldehyde. The binding ability of the fusion proteins to cell surface CD47 was analyzed using a flow cytometer (BD Calibur) (e.g., as shown in Figures 9A to 9C, Figures 9A to 9C show that the fusion proteins SS002 and SS002M91 specifically recognized the surface CD47 of Raji cells, Jurkat cells, and A549 cells, respectively).

[0133] The results showed that the fusion protein SS002M91 could specifically recognize CD47 on Raji, Jurkat, and A549 cells, and the recognition activity was significantly higher than that of SS002 and was dose-dependent. The EC50 values ​​for binding to Raji cells were 197.0 ng / mL for SS002M91 and 1140.0 ng / mL for SS002 (e.g., shown in Figure 9A). The EC50 values ​​for binding to Jurkat cells were 796.0 ng / mL for SS002M91 and 4529.0 ng / mL for SS002 (e.g., shown in Figure 9B). The EC50 values ​​for binding to A549 cells were 321.9 ng / mL for SS002M91 and 1655.0 ng / mL for SS002 (e.g., shown in Figure 9C).

[0134] Similarly, using the above-described methods described in this Example, the activities of SS002M91 and TTI-621 in recognizing CD47 molecules on the cell surface of Raji cells (e.g., as shown in Figure 10A), Jurkat cells (e.g., as shown in Figure 10B), and A549 cells (e.g., as shown in Figure 10C) were compared. The results showed that when binding to Raji cells, the maximum fluorescence intensity of SS002M91 was approximately 1200, and that of TTI-621 was approximately 600; when binding to Jurkat cells, the maximum fluorescence intensity of SS002M91 was approximately 5000, and that of TTI-621 was approximately 3000; when binding to A549 cells, the maximum fluorescence intensity of SS002M91 was approximately 180, and that of TTI-621 was approximately 60. Therefore, the maximum fluorescence intensity of SS002M91, which specifically recognizes CD47 molecules on the surface of tumor cells, was clearly higher than that of TTI-621 when binding to Raji cells. The EC50 values ​​for SS002M91 and TTI-621 were 13.06 ng / mL and 40.37 ng / mL, respectively, for binding to Jurkat cells, 28.09 ng / mL and 53.92 ng / mL, respectively, and for binding to A549 cells, 26.95 ng / mL and 1003 ng / mL, respectively, demonstrating that the half-effective dose of SS002M91 specifically recognizes CD47 molecules on tumor cell surfaces, demonstrating that SS002M91 is significantly superior to TTI-621 in specifically recognizing CD47 molecules on tumor cell surfaces.

[0135] Example 8 Blood coagulation reaction measurement Using SS002 and SS002M91 as examples, blood coagulation activity analysis was performed using the CD47 antibody Hu5F9-G4 (see Guerriero JL, Sotayo A, Ponichtera HE, et al. Class IIa HDAC inhibition reduces breast tumors and metastases through anti-tumor macrophages. [J]. Nature, 2017, 543(7645):428-432 and Gholamin S, Mitra SS, Feroze AH et al. Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors. Sci. Transl. Med 2017) as a control.

[0136] Human red blood cells were prepared from whole blood obtained from healthy donors (peripheral blood collected from volunteers). The whole blood was diluted 5-fold with PBS and washed three times to prepare a fresh 1% red blood cell solution. 50 μL of different concentrations of SIRPα fusion proteins SS002 and SS002M91 and anti-CD47 antibody Hu5F9-G4 were added to each well of a blood coagulation plate. 50 μL of 1% red blood cell solution was added to each well, mixed gently, and incubated overnight at 37°C and 5% CO2. The plate was then imaged to determine whether the red blood cells were fully agglutinated, sinking to the bottom of the well, and forming a flat mesh-like structure (100% agglutination, ++++). If the red blood cells were fully agglutinated and formed dots, a negative (-) was used to indicate non-agglutination (Figure 11).

[0137] The results in Figure 11 showed that the fusion proteins SS002M91 and SS002 did not induce hemagglutination, while the CD47 antibody Hu5F9-G4 could significantly induce hemagglutination within a certain dose range.

[0138] Example 9 In vivo antitumor activity measurement Using the fusion protein SS002M91 as an example, in vivo antitumor activity analysis was performed.

[0139] A tumor model was established by inoculating Raji-Luc cells into B-NSG mice to evaluate the anti-tumor activity of the SS002M91 antibody. Eight-week-old female B-NSG mice (Beijing Baiaosaitu Gene Biotechnology Co., Ltd.) were used as experimental animals, and Raji-Luc cells (Beijing Baiaosaitu Gene Biotechnology Co., Ltd.) were used to measure the tumor activity. Raji-Luc cells were transfected with a luciferase reporter gene to obtain a stable cell line, which was then resuscitated to the desired number. Logarithmic-phase cells were harvested and cultured at 5 x 10 6 The suspension was suspended at a concentration of 0.2 mL / 0.2 cells / 10 cells / 0.2 mL and inoculated into B-NSG mice via the tail vein at 0.2 mL / mouse. Tumor growth and body weight were observed using a small animal imager on days 0 and 3 after inoculation. On day 3, tumor imaging signals were observed at a moderate level (approximately 1.00 × 10 cells / 0.2 mL). 6 Twelve mice (P / S) were randomly assigned to two groups of six each: a vehicle control group (G1, saline) and an experimental group (G2, SS002M91) receiving 10 mg / kg of SS002M91. The mice were administered 10 mg / kg twice, once on the day of assignment and once on the third day. Tumor growth and survival were monitored (Figures 12A and 12B, respectively).

[0140] The results showed that on the 10th day after group administration, the mean tumor fluorescence intensity in the control group was 6.75 × 10 8 P / S, tumor mean fluorescence intensity in the treatment group was 1.76 × 10 6 P / S, indicating that the inhibition rate was approximately 95%.

[0141] Example 10: Measurement of effects on red blood cells and platelets Using the fusion protein SS002M91 as an example, preliminary in vivo safety evaluation was performed using B-NSG mice as a model.

[0142] Eighteen 8-week-old female B-NSG mice (Beijing baiaosaitu gene Biotechnology Co., Ltd.) were randomly divided into three groups of six mice each: the vehicle control group (administered saline), the experimental group (administered the fusion protein SS002M91), and the positive control group (administered TTI-621). The mice were administered 10 mg / kg of TTI-621 three times: on the day of grouping, the third day, and the seventh day. The red blood cell and platelet contents in the mouse peripheral blood were analyzed on the day after the third administration (i.e., the eighth day) (Figures 13A and 13B, respectively).

[0143] The results showed that compared with the control group, SS002M91 did not induce red blood cells (P=0.4483) but significantly reduced platelets (P=0.9199), and TTI-621 reduced red blood cells (P=0.0246) even though it had a small effect on platelets (P=0.9447).

[0144] Example 11 Effect of fusion protein activity on different subtype IgG Fc fusion According to the fusion protein construction method in Example 2, the SIRPα domain mutants M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111, M122, M126, M130, M135, and M145 obtained in Example 1 were each fused with human IgG4-Fc (the amino acid sequence of which is shown in SEQ ID NO: 68) and expressed to obtain the corresponding truncated domain of SIRPα mutant 1-human Fc fusion proteins (abbreviated as fusion proteins). These fusion proteins were designated as SS002M1G4, SS002M5G4, SS002M12G4, SS002M35G4, SS002M37G4, SS002M41G4, SS002M57G4, SS002M60G4, SS002M70G4, SS002M80G4, SS002M90G4, SS002M10G4, SS002M11G4, SS002M12G4, SS002M35G4, SS002M37G4, SS002M41G4, SS002M57G4, SS002M13G4, SS002M14G4, SS002M15G4, SS002M16G4, SS002M17G4, SS002M18G4, SS002M19G4, SS002M20G4, SS002M21G4, SS002M22G4, SS002M23G4, SS002M24G4 and SS002M67G4, SS002M81G4, SS002M82G4, SS002M84G4, SS002M91G4, SS002M99G4, SS002M102G4, SS002M111G4, SS002M122G4, SS002M126G4, SS002M130G4, SS002M135G4 and SS002M145G4 (the amino acid sequences of which are represented by SEQ ID NOs: 41-60, respectively).

[0145] As an example, the fusion protein SS002M91G4 was selected and subjected to bioactivity analysis.

[0146] The binding activity of SS002M91G4 to the antigen CD47 was analyzed using the binding activity measurement method in Example 2, and the results are shown in Figure 14. The results in Figure 14 show that SS002M91G4 has good CD47 antigen binding activity, with an EC50 value of 0.0157 μg / mL, which is nearly identical to the EC50 of SS002M91 (0.0195 μg / mL).

[0147] According to the specific analysis method for blocking CD47 / SIRPα interaction in Example 6, the blocking activity of SS002M91G4 against CD47 / SIRPα interaction was analyzed, and the results are shown in Figure 15. The results in Figure 15 show that SS002M91G4 has good blocking activity against CD47 / SIRPα interaction, with an IC50 value of 3.46 μg / mL, which is almost identical to the IC50 value of SS002M91 (5.47 μg / mL).

[0148] These results demonstrate that the fusion of different subtype IgGFc did not significantly affect the activity of the fusion protein constructed according to the present invention.

[0149] Example 12 Measurement of blood coagulation reaction of fusion protein Using SS002M91G4 as an example, the blood coagulation response of the above IgG4 Fc-based fusion proteins was evaluated with reference to the blood coagulation response assay method in Example 8. Human red blood cells were prepared from whole blood from healthy donors. The whole blood was diluted 5-fold with phosphate buffered saline (PBS) and washed three times to prepare a fresh 1% red blood cell solution. 50 μL of different concentrations of the fusion protein SS002M91G4, the positive control TTI-621, and the anti-CD47 antibody Hu5F9-G4 were added to each well of a blood coagulation plate. 50 μL of 1% red blood cell solution was also added to each well, mixed gently, and incubated overnight at 37°C and 5% CO2. After overnight incubation, the plate was imaged. The reading standard was 100% agglutination (++++) when all the red blood cells agglutinated, sank to the bottom of the hole, and spread out to form a flat mesh-like structure, and non-agglutination (-) when all the red blood cells sank to the bottom of the hole and formed dots.

[0150] The results are shown in Figure 16. The results showed that the IgG4 Fc-based fusion protein SS002M91G4 did not induce hemagglutination with TTI-621, while the CD47 antibody Hu5F9-G4 significantly induced hemagglutination within a certain dose range.

[0151] Example 13 Analysis of fusion protein biological activity Taking SS002M91G4 as an example, the Jurkat-CSR cell line (ImmuneOnco Biopharmaceuticals (Shanghai) Co., Ltd.) was used to evaluate the biological activity of the above IgG4 Fc-based fusion proteins with TTI-621 as a positive control.

[0152] CD47-Fc protein (Cat#12283-H02H, Sino Biological) was diluted to 0.2 μg / mL, and 50 μL of the diluted protein solution was added to each well. TTI-621 and SS002M91G4 were also diluted to 0.4 mg / mL, and then gradient diluted to different concentrations. 50 μL of each solution was added to each well. The cells were incubated with CD47-Fc for 45 min in a culture incubator at 37°C and 5% CO2. Jurkat-CSR cells were harvested and incubated at a density of 5 × 10 5 The cell suspension was adjusted to 1 / mL, and 100 μL of the cell suspension was added to each well. At the same time, a blank control group was placed and co-cultured with the protein mixture in a culture incubator at 37°C and 5% CO2 for 20 hours. After incubation, 20 μL of CCK-8 (Dojindo, Dojindo Molecular Technologies, Inc.) was added to each well, and the cells were cultured in a culture incubator at 37°C and 5% CO2 for 4 hours. The OD value at 450 nm was measured using a microplate reader, and the cell growth inhibition rate was calculated using the following formula. Inhibition rate % = (OD450 (sample) - OD450 (blank) / (OD450 (Jurkat-CSR) - OD450 (blank)) x 100

[0153] According to the principle of Jurkat-CSR cell lineage, CD47 / SIRPα interaction can induce target cell apoptosis, and the apoptosis signal can be blocked by adding an inhibitor that inhibits CD47 / SIRPα interaction. The stronger the inhibitor's effect, the more effectively the apoptosis signal is blocked. The results are shown in Figure 17. The results showed that SS002M91G4 can clearly block CD47-Fc-induced Jurkat-CSR cell apoptosis, and the blocking effect is stronger than that of TTI-621, and the IC 50 was approximately 1 / 100 of that of TTI-621, indicating that the biological activity of SS002M91G4 in blocking CD47 / SIRPα interaction is significantly superior to that of TTI-621.

[0154] Example 14 In vivo antitumor activity and effects on red blood cells and platelets of different IgG subtype Fc fusion proteins Using the fusion proteins SS002M91 and SS002M91G4 as examples, we analyzed their in vivo antitumor activity and the effects on red blood cells and platelets.

[0155] Raji cells were subcutaneously inoculated into NOD / SCID mice to establish a human lymphoma subcutaneous tumor model, and the in vivo antitumor activity of SS002M91 and SS002M91G4 was evaluated.

[0156] Six to seven week old female NOD / SCID mice (Shanghai Lingchang Biotechnology Co., Ltd.) were selected and Raji cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. 1 × 10 Raji cells were cultured in the logarithmic growth phase. 7 Raji cells were harvested, resuspended in PBS to an appropriate concentration, and plated on matrigel (BD Matrigel TM ) at a 1:1 ratio and used to inoculate subcutaneous tumors in mice. After inoculation, the average tumor volume was approximately 98.6 mm 3After that, the mice were randomly divided into four groups (i.e., vehicle control group, SS002M91 group, SS002M91G4 group, and TTI-621 group) according to tumor size, with each group consisting of six mice. The mice were intraperitoneally injected with 10 mg / kg twice a week for a total of two weeks (the four groups were administered PBS solution, SS002M91, SS002M91G4, and TTI-621, respectively).

[0157] Six days after the final administration, the experiment was completed. Blood samples were collected for routine blood tests to analyze the red blood cell and platelet contents in the mouse peripheral blood (red blood cell and platelet contents are shown in Figures 18A and 18B, respectively). During the administration period, tumor growth in the mice was monitored, and the therapeutic efficacy was evaluated based on the relative tumor growth inhibition rate (TGI) (results are shown in Figure 19). Safety was also evaluated based on changes in animal weight and mortality (results are shown in Figure 20). The formula for calculating TGI (%), i.e., the relative tumor growth inhibition rate, was TGI% = (1-T / C) × 100%, where T and C are the relative tumor volume (RTV) or tumor weight (TW), respectively, of the treatment group (e.g., the SS002M91 group, the SS002M91G4 group, and the TTI-621 group) and the control group (i.e., the vehicle control group) at a given time point.

[0158] The results showed that the SS002M91, SS002M91G4, and TTI-621 (10 mg / kg) groups all had significant tumor-inhibitory effects six days after drug discontinuation, with relative tumor growth inhibition rates (TGI) of 74.09%, 66.65%, and 54.75%, respectively, all of which were statistically significantly different from the vehicle control group (all p<0.01). The SS002M91 and SS002M91G4 groups showed similar tumor-inhibitory effects and were superior to the positive control TTI-621 group.

[0159] No animals died during the treatment period, no significant drug toxicity was observed, and the drug was well tolerated.Routine blood test results showed that the SS002M91 group had slightly decreased red blood cells and platelets compared with the positive control TTI-621 group, and the TTI-621 group also had a slightly decreased platelet count, but SS002M91G4 had no significant effect on red blood cells.

[0160] The above detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Those skilled in the art will recognize that there are many variations and modifications to the embodiments described hereinabove, which fall within the scope of the appended claims and the corresponding embodiments.

Claims

1. A human SIRPα domain mutant, the human SIRPα domain comprises amino acid residues 33 to 149 of human SIRPα variant 1, and the amino acid residues 33 to 149 (E33-S149) of human SIRPα variant 1 are set forth in SEQ ID NO: 63; The human SIRPα domain mutant comprises an amino acid mutation selected from the group consisting of: (1) I61V, E77N, Q82S, K83R and E84H; (2) I61F, V63I, K83R, E84K and V132I; (3) I61F, D95H, L96S, G109H, and V132S; (4) I61F, D95H, L96S, K98R, G109H, and V132S; (5) I61L, E77Q, E84D, V93A, R107N, and V132I; and (6) I61L, V63I, Q82G, E84G, D95R, L96S, N100D and V132I.

2. Contains the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 14; The human SIRPα domain mutant of claim 1.

3. A human SIRPα domain mutant according to any one of claims 1 to 2, Fusion proteins.

4. the fusion protein further comprises an immunoglobulin Fc region, and the human SIRPα domain mutant is directly or indirectly linked to the immunoglobulin Fc region. The fusion protein of claim 3.

5. The immunoglobulin Fc region has an IgG Fc region. The fusion protein of claim 4.

6. The IgG is selected from the group of IgG1 and / or IgG4, The fusion protein of claim 5.

7. the human SIRPα domain is located at the N-terminus of the immunoglobulin Fc region; The fusion protein according to any one of claims 4 to 6.

8. The immunoglobulin Fc region comprises an amino acid sequence represented by any one of SEQ ID NOs: 67-68. A fusion protein according to any one of claims 4 to 7.

9. containing the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 50, SEQ ID NO; 51, SEQ ID NO: 52 or SEQ ID NO: 54, A fusion protein according to any one of claims 3 to 8.

10. A gene encoding the human SIRPα domain mutant of any one of claims 1 to 2 or the fusion protein of any one of claims 3 to 9. Nucleic acid molecule.

11. 11. The nucleic acid molecule of claim 10. vector.

12. 12. A method for producing a nucleic acid molecule comprising the steps of: host cell.

13. Culturing the host cell of claim 12 under conditions that allow expression of the fusion protein. A method for preparing the fusion protein according to any one of claims 3 to 9.

14. A method for the preparation of a human SIRPα domain mutant according to any one of claims 1 to 2, a fusion protein according to any one of claims 3 to 9, a nucleic acid molecule according to claim 10, a vector according to claim 11 and / or a host cell according to claim 12, and optionally a pharmaceutically acceptable adjuvant. composition.

15. A human SIRPα domain mutant according to any one of claims 1 to 2, a fusion protein according to any one of claims 3 to 9, a nucleic acid molecule according to claim 10, a vector according to claim 11, a host cell according to claim 12 and / or a composition according to claim 14 for use in the prevention or treatment of tumors or autoimmune diseases.

16. The human SIRPα domain mutant, fusion protein, nucleic acid molecule, vector, host cell and / or composition of claim 15, wherein the tumor is selected from the group of CD47-positive hematological tumors and / or CD47-positive solid tumors.

17. The human SIRPα domain mutant, fusion protein, nucleic acid molecule, vector, host cell and / or composition of claim 15, wherein the autoimmune disease is selected from the group consisting of Crohn's disease, allergic asthma and / or rheumatoid arthritis.

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