Fusion protein comprising SIRPα mutant

Abstract

The present application relates to a bifunctional fusion protein, which comprises a first binding domain that specifically binds to CLDN18.2 and a second binding domain that specifically binds to the CD47 protein, wherein the second binding domain has at least one of the following characteristics: (a) binds to the CD47 protein with a K ‑8 value of 4×10 D M or lower; (b) specifically blocks the interaction between the CD47 protein and SIRPα; (c) does not cause a coagulation reaction; and (d) inhibits the growth and / or proliferation of tumors or tumor cells.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically relates to a multifunctional fusion protein that binds to CD47 and CLDN18.2, and its application in treating diseases and / or disorders. Background Art

[0002] CD47 protein is a transmembrane glycoprotein and belongs to the immunoglobulin superfamily. In addition to normal tissue cells expressing CD47, many tumor cells overexpress CD47. The binding of CD47 on the surface of tumor cells to SIRPα on the surface of macrophages will prevent macrophages from phagocytosing tumor cells, which is regarded as a mechanism for tumors to evade the body's immune surveillance. Blocking the interaction between CD47 protein and SIRPα can inhibit tumor growth.

[0003] Tight junction proteins are a family of tight junction membrane proteins expressed in epithelia and endothelia and forming paracellular barriers and micropores that determine the permeability of tight junctions. Claudin 18 isoform 2 (CLDN18.2), which is a splice variant of claudin 18 protein, is a gastric antigen expressed on short-lived differentiated gastric epithelial cells. Dysregulation of the expression of tight junction proteins can be detected in many cancers and may lead to tumorigenesis and cancer invasion. The expression of CLDN18.2 is elevated in pancreatic ductal adenocarcinoma, esophageal tumors, non-small cell lung cancer, ovarian cancer, cholangiocarcinoma, and cholangiocarcinoma. Summary of the Invention

[0004] The present application provides a bifunctional fusion protein, which comprises a portion specifically binding to CLDN18.2 and a portion specifically binding to CD47 protein. The fusion protein described in the present application can effectively inhibit the growth and / or proliferation of tumors or tumor cells.

[0005] On the one hand, the present application provides a fusion protein, which comprises: a first binding domain specifically binding to CLDN18.2; and a second binding domain specifically binding to CD47 protein; wherein the second binding domain has at least one of the following characteristics: (a) binding to CD47 protein with a K -8 value of 4×10 D M or lower; (b) specifically blocking the interaction between CD47 protein and SIRPα; (c) not causing a coagulation reaction; and (d) inhibiting the growth and / or proliferation of tumors or tumor cells.

[0006] In certain embodiments, the second binding domain comprises a mutant of human SIRPα variant 1, and the mutant contains amino acid residue mutations at one or more positions compared with the amino acid sequence from position 33 to position 149 of SEQ ID NO:22.

[0007] In certain embodiments, the mutant in the fusion protein comprises an amino acid mutation at one or more amino acid positions selected from the group consisting of: L44, I61, V63, E77, Q82, K83, E84, V93, D95, L96, K98, N100, R107, G109, and V132.

[0008] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the I61 and E77 amino acid residues.

[0009] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the I61 and V132 amino acid residues.

[0010] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the I61 and E84 amino acid residues.

[0011] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the I61, K83, and E84 amino acid residues.

[0012] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the I61, G109, and V132 amino acid residues.

[0013] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the I61, D95, L96, G109, and V132 amino acid residues.

[0014] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the V93 and R107 amino acid residues.

[0015] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the L44 and I61 amino acid residues.

[0016] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the L44 and E77 amino acid residues.

[0017] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the Q82, K83, and E84 amino acid residues.

[0018] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions at the E77 and V132 amino acid residues.

[0019] In certain embodiments, the mutant in the fusion protein comprises an amino acid mutation at an amino acid residue selected from the group consisting of:

[0020] (1) I61, V63, E77, E84, V93, L96, K98, N100 and V132;

[0021] (2) I61, E77, Q82, K83 and E84;

[0022] (3) I61, V63, K83, E84 and V132;

[0023] (4) I61, E77, E84, R107 and V132;

[0024] (5) I61, V63, E77, K83, E84 and N100;

[0025] (6) I61, E77, Q82, K83, E84 and R107;

[0026] (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109 and V132;

[0027] (8) I61, E77, Q82, K83, E84 and V132;

[0028] (9) I61;

[0029] (10) I61, D95, L96, G109 and V132;

[0030] (11) I61, D95, L96, K98, G109 and V132;

[0031] (12) I61, E77, E84, V93, R107 and V132;

[0032] (13) E77, L96, N100, G109 and V132;

[0033] (14) I61, V63, Q82, E84, D95, L96, N100 and V132;

[0034] (15) I61, E77, Q82, K83, E84, V93, D95, L96, K98, N100 and V132;

[0035] (16) I61, E77, Q82, K83, E84 and V93;

[0036] (17) I61, V63, E77, K83, E84, D95, L96, K98 and N100;

[0037] (18) I61, V63, E77, K83, D95, L96, K98, N100, and G109;

[0038] (19) I61, E77, Q82, E84, V93, D95, L96, K98, and N100;

[0039] (20) I61, V63, E77, Q82, and E84; and

[0040] (21) L44, I61, E77, Q82, K83, E84, and V132.

[0041] In certain embodiments, the mutant in the fusion protein comprises one or more amino acid substitutions selected from the group consisting of: L44V, I61L / V / F, V63I, E77I / N / Q / K / H / M / R / V / L, Q82S / R / G / N, K83R, E84Q / K / 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.

[0042] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions I61L / V / F and E77Q / N / I.

[0043] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions I61L / V / F and V132I / S.

[0044] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions I61L / V / F, K83R, and E84Q / D / H.

[0045] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions I61L / V / F, G109H, and V132I / S.

[0046] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions I61L / V / F, D95H, L96S, G109H, and V132I / S.

[0047] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions V93A and R107N.

[0048] In certain embodiments, the mutant in the fusion protein comprises the amino acid substitutions L44V and I61L / V / F.

[0049] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions of L44V and E77Q / N / I.

[0050] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions of Q82S / R / G / N, K83R, and E84Q / K / H / D / R / G.

[0051] In certain embodiments, the mutant in the fusion protein comprises amino acid substitutions of E77Q / N / I and V132I / S.

[0052] In certain embodiments, the mutant in the fusion protein comprises amino acid mutations selected from the group consisting of:

[0053] (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G, and V132L;

[0054] (2) I61V, E77N, Q82S, K83R, and E84H;

[0055] (3) I61F, V63I, K83R, E84K, and V132I;

[0056] (4) I61L, E77Q, E84D, R107N, and V132I;

[0057] (5) I61L, V63I, E77K, K83R, E84D, and N100G;

[0058] (6) I61V, E77H, Q82R, K83R, E84H, and R107S;

[0059] (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R, and V132R;

[0060] (8) I61L, E77M, Q82G, K83R, E84D, and V132L;

[0061] (9) I61L;

[0062] (10) I61F, D95H, L96S, G109H, and V132S;

[0063] (11) I61F, D95H, L96S, K98R, G109H, and V132S;

[0064] (12) I61L, E77Q, E84D, V93A, R107N, and V132I;

[0065] (13) E77K, L96S, N100K, G109H and V132L;

[0066] (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D and V132I;

[0067] (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D and

[0068] V132L;

[0069] (16) I61V, E77N, Q82S, K83R, E84H and V93A;

[0070] (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R and N100E;

[0071] (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D and G109R;

[0072] (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R and N100G;

[0073] (20) I61L, V63I, E77N, Q82G and E84G; and

[0074] (21) L44V, I61F, E77I, Q82R, K83R, E84Q and V132I.

[0075] In certain embodiments, the mutant in the fusion protein further comprises modification of one or more glycosylation sites.

[0076] In certain embodiments, the modification of the glycosylation site comprises a glycosylation site mutation.

[0077] In certain embodiments, the glycosylation site mutation is located at the N110 position.

[0078] In certain embodiments, the glycosylation mutation comprises an amino acid substitution of N110A.

[0079] In certain embodiments, the mutant in the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 1-21.

[0080] In some embodiments, the first binding domain of the fusion protein comprises an antigen-binding protein targeting CLDN18.2.

[0081] In some embodiments, the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0082] In some embodiments, the antibody is selected from the group consisting of: monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody, and fully human antibody.

[0083] In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, Fab’, F(ab)2, dAb, isolated complementarity-determining region CDR, Fv, and scFv.

[0084] In some embodiments, the CLDN18.2 comprises human CLDN18.2.

[0085] In some embodiments, the first binding domain comprises at least one CDR in the heavy-chain variable region (VH), and the VH comprises the amino acid sequence shown in SEQ ID NO:24.

[0086] In some embodiments, the first binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:25.

[0087] In some embodiments, the first binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:26.

[0088] In some embodiments, the first binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27.

[0089] In some embodiments, the first binding domain comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:24.

[0090] In some embodiments, the first binding domain of the fusion protein comprises an immunoglobulin single variable domain.

[0091] In some embodiments, the immunoglobulin single variable domain comprises V H H.

[0092] In some embodiments, the first binding domain in the fusion protein comprises at least one CDR in V H H, and the V H H comprises the amino acid sequence shown in SEQ ID NO:24.

[0093] In some embodiments, the VH H comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0094] In certain embodiments, the V H H comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26.

[0095] In certain embodiments, the V H H comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0096] In certain embodiments, the V H H comprises the amino acid sequence shown in SEQ ID NO: 24.

[0097] In certain embodiments, the first binding domain comprises a heavy chain constant region Fc fragment.

[0098] In certain embodiments, the heavy chain constant region is derived from IgG.

[0099] In certain embodiments, the heavy chain constant region is derived from human IgG.

[0100] In certain embodiments, the IgG is selected from the group consisting of IgG1 and IgG4.

[0101] In certain embodiments, the heavy chain constant region Fc fragment comprises the amino acid sequence of SEQ ID NO: 28.

[0102] In certain embodiments, the first binding domain of the fusion protein is located at the N-terminus of the second binding domain.

[0103] In certain embodiments, the fusion protein further comprises a linker, which is located at the C-terminus of the first binding domain and at the N-terminus of the second binding domain.

[0104] In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 24.

[0105] In certain embodiments, the fusion protein comprises at least two of the second binding domains.

[0106] In certain embodiments, each of the second binding domains of the fusion protein is located at the C-terminus of the first binding domain, respectively.

[0107] In certain embodiments, the fusion protein comprises at least one polypeptide chain, wherein the polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO: 30 - 35.

[0108] In certain embodiments, the fusion protein comprises two polypeptide chains, and each polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO: 30-35.

[0109] On the other hand, the present application also provides an immunoconjugate comprising the fusion protein.

[0110] On the other hand, the present application also provides one or more nucleic acid molecules encoding the fusion protein or the immunoconjugate.

[0111] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0112] On the other hand, the present application also provides a pharmaceutical composition comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, and optionally a pharmaceutically acceptable carrier.

[0113] On the other hand, the present application also provides a cell comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, or the vector.

[0114] On the other hand, the present application also provides a method for preparing the fusion protein, which comprises culturing the cell under conditions enabling the expression of the fusion protein.

[0115] On the other hand, the present application also provides the use of the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the pharmaceutical composition, and the cell in the preparation of a drug for preventing and / or treating a disease and / or disorder.

[0116] In certain embodiments, the disease and / or disorder comprises a disease and / or disorder related to CLDN18.2.

[0117] In certain embodiments, the disease and / or disorder related to CLDN18.2 includes a disease involving cells expressing CLDN18.2 or a disease related to cells expressing CLDN18.2.

[0118] In certain embodiments, the disease and / or disorder includes a tumor.

[0119] In certain embodiments, the tumor includes a solid tumor and / or a non-solid tumor.

[0120] In certain embodiments, the tumor includes gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and / or gallbladder cancer.

[0121] Those skilled in the art can easily insight into other aspects and advantages of this application from the following detailed description. Only the exemplary embodiments of this application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Accordingly, the descriptions in the drawings and the specification of this application are merely exemplary and not restrictive. Brief Description of the Drawings

[0122] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0123] Figure 1 It shows an example of the structure of the fusion protein described in this application.

[0124] Figure 2 It shows the binding result of the fusion protein described in this application with CD47.

[0125] Figures 3A - 3B It shows the binding result of the fusion protein described in this application with Raji cells.

[0126] Figure 4 It shows the binding result of the fusion protein described in this application with 293T / CLDN18.2 cells.

[0127] Figure 5 It shows the result of the fusion protein described in this application blocking the CD47 / SIRPα interaction.

[0128] Figure 6 It shows the ADCC activity detection of the fusion protein described in this application.

[0129] Figures 7A - 7B It shows the detection of the binding activity of the fusion protein described in this application with red blood cells.

[0130] Figure 8 It shows the growth curve of the mouse 293T / CLDN18.2 cell xenograft model after the start of treatment. Detailed Embodiments

[0131] The following specific embodiments illustrate the implementation manners of the invention of this application. Those skilled in the art can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0132] Term Definitions

[0133] In the present application, the term "fusion protein" generally refers to a polypeptide or protein composed of two or more moieties joined together. Each of these moieties can be a polypeptide or protein with different properties. Such properties can be biological properties, such as in vitro or in vivo activities. The property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. These two moieties can be directly linked by a single peptide bond or through a peptide linker. Fusion proteins can be artificially prepared by recombinant DNA technology. For example, 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 a fused nucleic acid molecule, which can encode the fusion protein. Translation of the fusion gene can produce a single polypeptide, which can have the properties of at least one, and even each, of the two or more proteins or polypeptides before fusion.

[0134] In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can determine the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) can be an antibody that binds to the target with a greater affinity, avidity, more easily, and / or for a longer duration than it binds to other targets. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding includes, but does not require, exclusive binding.

[0135] In the present application, the term "binding domain" generally refers to a domain that can specifically bind to and / or recognize a specific epitope on a target (such as an antigen). In the present application, the term "domain" generally refers to a distinct and compact globular structural region in the protein subunit structure. For example, a polypeptide chain can first form a regular secondary structure in certain regions where adjacent amino acid residues are arranged in a specific pattern. Then, adjacent secondary structure segments can be assembled together to form a super-secondary structure. On this basis, the polypeptide chain can fold into an approximately globular tertiary structure. For larger protein molecules or subunits, the polypeptide chain often consists of two or more spatially distinguishable and relatively independent regional structures associated to form a tertiary structure, and such relatively independent regional structures can be referred to as domains.

[0136] In the present application, the "first" and "second" in the terms "first binding domain" and "second binding domain" are only for descriptive distinction.

[0137] In the present application, the term "CD47 protein" generally refers to integrin-associated protein (IAP), which is a multi-transmembrane receptor belonging to the immunoglobulin superfamily. For example, the CD47 protein can bind to membrane integrins and bind to its ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). The CD47 protein is widely expressed on the cell membrane surface. In the present application, the CD47 protein may include any variants, isotypes, and species homologs of human CD47. The amino acid sequence of the human CD47 protein is listed as CEJ95640.1 in GenBank. The CD47 protein can be naturally expressed by cells or expressed on cells transfected with the CD47 gene.

[0138] In the present application, the term "SIRPα" generally refers to a regulatory membrane glycoprotein from the SIRP family, which can serve as a ligand for the CD47 protein. In the present application, the SIRPα may include human SIRPα.

[0139] In the present application, the term "human SIRPα domain" generally refers to the wild-type, endogenous mature form of human SIRPα, its fragments, or functional variants. In humans, two main forms of the SIRPα protein are found. The amino acid sequence of one form (variant 1 or type V1) is listed as NCBI RefSeq NP_542970.1 (residues 31 - 504 constitute the mature form). The other form (variant 2 or type V2) differs from the variant 1 or type V1 by 13 amino acids and the amino acid sequence is listed as CAA71403.1 in GenBank. These two forms of SIRPα constitute approximately 80% of the various types of SIRPα present in humans. In the present application, the term "antigen-binding protein" generally refers to a protein that has antigen-binding ability and is removed from its natural state. The "isolated antigen-binding protein" may include an antigen-binding portion and optionally, a framework or scaffold portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding portion to the antigen. The antigen-binding protein may include, for example, a protein framework region (FR) of antibody origin or an alternative protein framework region or an artificial framework region with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions containing, for example, biocompatible polymers.

[0140] In the present application, the term "CDR", also known as "complementary determining region", generally refers to the regions in the variable domains of antibodies, the sequences of which are highly variable and / or form structurally defined loops. Generally, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, considering Kabat / Chothia comprehensively, etc. These coding systems are known in the art, and for details, see, e.g., http: / / www.bioinf.org.uk / abs / index.html#kabatnum.

[0141] In the present application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a part of the heavy chain and / or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "V H " and "V L " (or "VH" and "VL" respectively). These domains are generally the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0142] In the present application, the term "antibody" generally refers to an immunoglobulin or its fragment or its derivative, covering any polypeptide including an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", such as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specifically bind to CLDN18.2).

[0143] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind to an antigen (e.g., CLDN18.2). In the present application, the antigen-binding fragment may include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, and / or dAb.

[0144] In the present application, the term "Fab" generally refers to the antigen-binding fragment of an antibody. As described above, intact antibodies can be digested with papain. After digestion of an antibody with papain, two identical antigen-binding fragments, i.e., "Fab" fragments, and a residual "Fc" fragment (i.e., the Fc region, as above) are produced. A Fab fragment can consist of a complete L chain and the variable region of one heavy chain and the first constant region (CH1) of that H chain (VH). In the present application, the term "Fab' fragment" generally refers to the monovalent antigen-binding fragment of a human monoclonal antibody. For example, a Fab' fragment can include all of the light chain, all of the heavy chain variable region, and all or part of the first and second constant regions of the heavy chain. For example, a Fab' fragment can also include part or all of the heavy chain. In the present application, the term "F(ab')2" generally refers to an antibody fragment produced by pepsin digestion of an intact antibody. The F(ab')2 fragment contains two Fab fragments held together by disulfide bonds and a portion of the hinge region. The F(ab')2 fragment has bivalent antigen-binding activity and is capable of cross-linking antigens. In the present application, the term "Fv fragment" generally refers to the monovalent antigen-binding fragment of a human monoclonal antibody, including all or part of the heavy chain variable region and the light chain variable region, and lacking the heavy chain constant region and the light chain constant region. The heavy chain variable region and the light chain variable region include, for example, CDRs. In the present application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment including the variable region of a light chain and at least one antibody fragment including the variable region of a heavy chain, wherein the light chain and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker), and capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used in the present application, the scFv can have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), the scFv can include VL-linker-VH or can include VH-linker-VL. In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, referring, for example, to Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6), referring to Holt et al., Trends Biotechnol., 2003, 21(11): 484-490; and referring, for example, to WO 06 / 030220, WO 06 / 003388, and other published patent applications of Domantis Ltd. The term "dAb" generally includes sdAb. The term "sdAb" generally refers to a single-domain antibody. A single-domain antibody generally refers to an antibody fragment consisting only of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL), and in some cases, a single-domain antibody can also include a heavy chain constant region.

[0145] In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules composed of a single molecule. Monoclonal antibodies are typically highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which generally have different antibodies against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, the monoclonal antibodies used in the present application can be prepared in hybridoma cells or can be prepared by recombinant DNA methods.

[0146] In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Typically, the variable region is derived from an antibody of an experimental animal such as a rodent ("parent antibody"), and the constant region is derived from a human antibody, such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent antibody.

[0147] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody are replaced with the corresponding amino acids derived from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions, or modifications of amino acids may also be permitted, provided that they still retain the ability of the antibody to bind to a specific antigen. A humanized antibody may optionally contain at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains the antigen specificity similar to the original antibody. The "humanized" form of a non-human antibody can minimally comprise a chimeric antibody with sequences derived from a non-human immunoglobulin. In some cases, the CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with the CDR region residues of a non-human species (donor antibody) having the desired properties, affinity, and / or ability (such as a llama, mouse, rat, rabbit, or non-human primate). In some cases, the FR region residues of a human immunoglobulin can be replaced with the corresponding non-human residues. In addition, a humanized antibody may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve the performance of the antibody, such as binding affinity.

[0148] The proteins, polypeptides, and / or amino acid sequences involved in the present application should also be understood to include at least the following scope: variants or homologs having the same or similar functions as the said protein or polypeptide.

[0149] In the present application, 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 the polypeptide (e.g., an antibody or fragment thereof that specifically binds CLDN18.2). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by substitution, deletion and / or insertion of at least 1, e.g., 1 - 30, 1 - 20 or 1 - 10, and further e.g., 1, 2, 3, 4 or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide prior to the change. For example, the substitution may be a conservative substitution.

[0150] In the present application, the homolog may be a protein or polypeptide having at least about 85% (e.g., having 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 higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds CLDN18.2).

[0151] In the present application, the homology generally refers to the similarity, analogy or correlation between two or more sequences. The "percent sequence homology" can be calculated by the following method: comparing two sequences to be aligned in a comparison window, determining the number of positions where the same nucleic acid bases (e.g., A, T, C, G, I) or the same 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) exist in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to generate the percent sequence homology. The alignment for determining 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. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment within the full-length sequence range being compared or within the target sequence region. The homology can also be determined by the following methods: FASTA and BLAST. The description of the FASTA algorithm can be found in "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and "Rapid and Sensitive Protein Similarity Searches" by D.J. Lipman and W.R. Pearson, Science, 227: 1435-1441, 1989. The description of the BLAST algorithm can be found in "Basic Local Alignment Search Tool" by S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, Journal of Molecular Biology, 215: 403-410, 1990.

[0152] In the present application, the terms "CLDN18.2" or "Claudin18.2" can be used interchangeably and generally refer to subtype 2 of the tight junction protein Claudin18. The terms cover "full-length", unprocessed CLDN18.2 and any form of CLDN18.2 produced by cell processing. CLDN18.2 can include the complete CLDN18.2 and its fragments, its functional variants, isoforms, species homologs, derivatives, analogs, and analogs having at least one common epitope with CLDN18.2.

[0153] Under normal circumstances, in a polypeptide chain, the connection of an amino group to another carboxyl group in the polypeptide chain can make it form a chain. However, at the two ends of a protein, there are respectively amino acid residues that have not formed peptide bonds, namely the end of the polypeptide chain carrying a free amino group and the end of the polypeptide chain carrying a carboxyl group. In this application, the term "N-terminus" generally refers to the end of a polypeptide chain where the amino acid residue carries a free amino group. In this application, the term "C-terminus" generally refers to the end of a polypeptide chain where the amino acid residue carries a free carboxyl group.

[0154] In this application, the term "nucleic acid molecule" generally refers to any length of isolated form of nucleotides, deoxyribonucleotides, ribonucleotides or their analogs that are isolated from their natural environment or artificially synthesized.

[0155] In this application, the term "immunoconjugate" generally refers to an immunologically functional polypeptide molecule conjugated with one or more heterologous molecules (including but not limited to cytotoxins). In this application, "conjugated", "linked" and "fused" can be used interchangeably in this application and generally refer to connecting two or more chemical elements, sequences or components together, such as by including chemical conjugation or recombinant means. The heterologous molecules can be cytotoxins, chemotherapeutic drugs, etc. For example, conjugating one or more heterologous molecules (such as cytotoxins) to the fusion protein described in this application can obtain the immunoconjugate described.

[0156] In the present invention, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. A vector can be transformed, transduced or transfected into a host cell so that the genetic material element it carries can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage and animal viruses, etc. 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, papillomaviruses, papovaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector may also contain an origin of replication. A vector may also include components to assist its entry into cells, such as virus particles, liposomes or protein coats, but not only these substances.

[0157] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or disorder. The pharmaceutical composition may comprise the isolated antigen-binding protein described in the present application, the nucleic acid molecule described in the present application, the vector and / or the cell described in the present application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the doses and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0158] In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals exposed thereto at the doses and concentrations employed.

[0159] In the present application, the term "tumor" generally refers to a neoplasm formed by the hyperplasia of local tissue cells in a mammalian body (e.g., cells or their components) under the action of various tumorigenic factors. In the present application, tumors may include solid tumors and non-solid tumors. In the present application, the tumors may comprise gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and / or gallbladder cancer.

[0160] In the present application, the term "comprising" generally means including the expressly designated features, but not excluding other elements.

[0161] In the present application, the term "about" generally means varying within a range of 0.5% - 10% above or below the specified value, e.g., varying within a range of 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 specified value. Detailed Description of the Invention

[0163] Fusion protein

[0164] On the one hand, the present application provides a fusion protein, which may comprise a first binding domain and a second binding domain. The first binding domain may specifically bind to CLDN18.2; the second binding domain may specifically bind to the CD47 protein, wherein the second binding domain may have at least one of the following characteristics: (a) with a K -8 M or lower Dbinds to the CD47 protein; (b) specifically blocks the interaction between the CD47 protein and SIRPα; (c) does not cause a coagulation reaction; and (d) can inhibit the growth and / or proliferation of tumors or tumor cells.

[0165] In certain embodiments, the second binding domain may comprise a mutant of SIRPα variant 1 or a fragment thereof. Compared with the amino acid sequence from position 33 to position 149 of SEQ ID NO:22, the mutant comprises mutations of amino acid residues at one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) positions. The amino acid residue mutations may comprise substitutions, deletions or additions of amino acid residues. The positions of the amino acids are determined by the amino acid positions shown in SEQ ID NO:22. For example, I61 generally refers to the 61st amino acid based on the amino acid sequence shown in SEQ ID NO:22. In the present application, the amino acid sequence shown in SEQ ID NO:23 may refer to the amino acid residues from position 33 to position 149 of human SIRPα variant 1 (SEQ ID NO:22), that is, the position number of the first amino acid of the amino acid sequence shown in SEQ ID NO:23 is "position 33".

[0166] In the present application, the mutant of SIRPα variant 1 may include one or more amino acid mutations in the amino acid sequence from position 33 to position 149 of SIRPα variant 1. For example, the mutant of SIRPα variant 1 may comprise one or more amino acid mutations in the amino acid sequence shown in SEQ ID NO:23. For example, the mutant of SIRPα variant 1 described in the present application comprises one or more amino acid mutations compared with the amino acid sequence shown in SEQ ID NO:23.

[0167] In the present application, the fusion protein can bind to both the tumor-associated antigen and the CD47 protein, thereby playing a role in treating diseases and / or disorders.

[0168] In the present application, the term "first binding domain" generally refers to a domain that can specifically bind to CLDN18.2. The term "second binding domain" generally refers to a domain that can specifically bind to the CD47 protein.

[0169] Second binding domain that specifically binds to CD47

[0170] In the present application, the mutant (e.g., a mutant of human SIRPα variant 1 that specifically binds to the CD47 protein) contains an amino acid substitution at one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid residues selected from the group consisting of: L44, I61, V63, E77, Q82, K83, E84, V93, D95, L96, K98, N100, R107, G109, and V132.

[0171] For example, the mutant may contain an amino acid mutation at position L44. For example, the mutant may contain an amino acid mutation at position I61. For example, the mutant may contain an amino acid mutation at position V63. For example, the mutant may contain an amino acid mutation at position E77. For example, the mutant may contain an amino acid mutation at position Q82. For example, the mutant may contain an amino acid mutation at position K83. For example, the mutant may contain an amino acid mutation at position E84. For example, the mutant may contain an amino acid mutation at position V93. For example, the mutant may contain an amino acid mutation at position D95. For example, the mutant may contain an amino acid mutation at position L96. For example, the mutant may contain an amino acid mutation at position K98. For example, the mutant may contain an amino acid mutation at position N100. For example, the mutant may contain an amino acid mutation at position R107. For example, the mutant may contain an amino acid mutation at position G109. For example, the mutant may contain an amino acid mutation at position V132.

[0172] In the present application, the position of the amino acid residue in the amino acid substitution is determined based on the residue numbering of the amino acid sequence shown in SEQ ID NO:22.

[0173] "Amino acid substitution Xn" means that an amino acid substitution occurs at the residue X at the nth position corresponding to the amino acid sequence shown in SEQ ID NO:22, where n is a positive integer and X is the abbreviation of any amino acid residue. For example, "amino acid substitution I61" means that an amino acid substitution occurs at the residue I at the 61st position corresponding to the amino acid sequence shown in SEQ ID NO:22.

[0174] In the present application, the amino acid substitution may be a non-conservative substitution. The non-conservative substitution may include changing the amino acid residue in the target protein or polypeptide in a non-conservative manner, for example, changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with a different side chain size or a different property (e.g., hydrophobicity).

[0175] In the present application, the amino acid substitution may also be a conservative substitution. The conservative substitution may include changing the amino acid residue in the target protein or polypeptide in a conservative form, for example, changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with the same or similar side chain size or the same or similar property (e.g., still hydrophilic). Such conservative substitutions generally do not have a great impact on the structure or function of the resulting protein. In the present application, the amino acid sequence variant of the fusion protein or its fragment may include conservative amino acid substitutions that do not significantly change the protein structure or its function (e.g., mutants that block the binding of CD47 to the human SIRPα variant 1 that specifically binds to the CD47 protein).

[0176] As an example, the mutual substitution between each amino acid within each of the following groups may be considered a conservative substitution in the present application: Amino acid group with non-polar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. Amino acid group with uncharged, polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Amino acid group with negatively charged, polar side chains: aspartic acid and glutamic acid. Positively charged basic amino acids: lysine, arginine, and histidine. Amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0177] In the present application, the mutant may comprise an amino acid substitution at an amino acid residue 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 V132; (13) E77, L96, N100, G109 and V132; (14) I61, V63, Q82, E84, D95, L96, N100 and V132; (15) I61, E77, Q82, K83, E84, V93, D95, L96, K98, N100 and V132; (16) I61, E77, Q82, K83, E84 and V93; (17) I61, V63, E77, K83, E84, D95, L96, K98 and N100; (18) I61, V63, E77, K83, D95, L96, K98, N100 and G109; (19) I61, E77, Q82, E84, V93, D95, L96, K98 and N100; (20) I61, V63, E77, Q82 and E84; and (21) L44, I61, E77, Q82, K83, E84 and V132.

[0178] In the present application, the mutant may comprise one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid substitutions selected from the group consisting of: L44V, I61L / V / F, V63I, E77I / N / Q / K / H / M / R / V / L, Q82S / R / G / N, K83R, E84Q / K / 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.

[0179] In the present application, the amino acid substitution "XnY / Z" means that the residue X at the nth position in the amino acid sequence shown in SEQ ID NO: 22 is substituted with the amino acid residue Y or the amino acid residue Z, where n is a positive integer, and X, Y, and Z are each independently abbreviations of any amino acid residue, and X is different from Y or Z. For example, the amino acid substitution "I61L / V / F" means that the residue I at the 61st position in the amino acid sequence shown in SEQ ID NO: 22 is substituted with the amino acid residue L, V, or F.

[0180] For example, the mutant may comprise the L44V amino acid mutation. For example, the mutant may comprise the I61L, I61V, or I61F amino acid mutations. For example, the mutant may comprise the V63I amino acid mutation. For example, the mutant may comprise the E77I, E77N, E77Q, E77K, E77H, E77M, E77R, E77V, or E77L amino acid mutations. For example, the mutant may comprise the Q82S, Q82R, Q82G, or Q82N amino acid mutations. For example, the mutant may comprise the K83R amino acid mutation. For example, the mutant may comprise the E84Q, E84K, E84H, E84D, E84R, or E84G amino acid mutations. For example, the mutant may comprise the V93L or V93A amino acid mutations. For example, the mutant may comprise the D95H, D95R, or D95E amino acid mutations. For example, the mutant may comprise the L96S or L96T amino acid mutations. For example, the mutant may comprise the K98R amino acid mutation. For example, the mutant may comprise the N100G, N100K, N100D, or N100E amino acid mutations. For example, the mutant may comprise the R107N or R107S amino acid mutations. For example, the mutant may comprise the G109R or G109H amino acid mutations. For example, the mutant may comprise the V132L, V132R, V132I, or V132S amino acid mutations.

[0181] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61 and E77. For example, the mutant may comprise the amino acid substitutions I61L / V / F and E77Q / N / I.

[0182] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61 and V132. For example, the mutant may comprise the amino acid substitutions I61L / V / F and V132I / S.

[0183] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61 and E84. For example, the mutant may comprise the amino acid substitutions I61L / V / F and E84D / H.

[0184] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61, K83, and E84. For example, the mutant may comprise amino acid substitutions of I61L / V / F, K83R, and E84Q / D / H.

[0185] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61, D95, and L96. For example, the mutant may comprise amino acid substitutions of I61L / V / F, D95H, and L96S.

[0186] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61, D95, L96, G109, and V132. For example, the mutant may comprise amino acid substitutions of I61L / V / F, D95H, L96S, G109H, and V132I / S.

[0187] In the present application, the mutant may comprise substitutions at the following amino acid positions: I61, G109, and V132. For example, the mutant may comprise amino acid substitutions of I61L / V / F, G109H, and V132I / S.

[0188] In the present application, the mutant may comprise substitutions at the following amino acid positions: V93 and R107. For example, the mutant may comprise amino acid substitutions of V93A and R107N.

[0189] In the present application, the mutant may comprise substitutions at the following amino acid positions: L44 and I61. For example, the mutant may comprise amino acid substitutions of L44V and I61L / V / F.

[0190] In the present application, the mutant may comprise substitutions at the following amino acid positions: L44 and E77. For example, the mutant may comprise amino acid substitutions of L44V and E77Q / N / I.

[0191] In the present application, the mutant may comprise substitutions at the following amino acid positions: Q82, K83, and E84. For example, the mutant may comprise amino acid substitutions of Q82S / R / G / N, K83R, and E84Q / K / H / D / R / G.

[0192] In the present application, the mutant may comprise substitutions at the following amino acid positions; E77 and V132. For example, the mutant may comprise amino acid substitutions of E77Q / N / I and V132I / S.

[0193] In the present application, the mutant may comprise an amino acid substitution selected from the group consisting of: (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G and V132L; (2) I61V, E77N, Q82S, K83R and E84H; (3) I61F, V63I, K83R, E84K and V132I; (4) I61L, E77Q, E84D, R107N and V132I; (5) I61L, V63I, E77K, K83R, E84D and N100G; (6) I61V, E77H, Q82R, K83R, E84H and R107S; (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R and V132R; (8) I61L, E77M, Q82G, K83R, E84D and V132L; (9) I61L; (10) I61F, 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; (20) I61L, V63I, E77N, Q82G and E84G; and (21) L44V, I61F, E77I, Q82R, K83R, E84Q and V132I.

[0194] In the present application, based on the truncated domain of human SIRPα variant 1 (the amino acid sequence shown in SEQ ID NO: 23, i.e., residues 33 - 149 in the amino acid sequence of human SIRPα variant 1), mutants of SIRPα variant 1 respectively containing the amino acid substitution groups of (1) - (21) above can be named M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111, M122, M126, M130, M135, M145, and M169 in sequence.

[0195] In the present application, the mutant may further comprise modification of one or more glycosylation sites. The modification comprises changing the glycosylation sites by artificial means. For example, the modification of glycosylation sites comprises glycosylation site mutations. In some embodiments, the glycosylation site mutation is located at position N110. In some embodiments, the glycosylation site mutation comprises N110A.

[0196] In the present application, compared with the amino acid sequence shown in SEQ ID NO: 23, the mutant (e.g., the mutant of human SIRPα variant 1 that specifically binds to CD47 protein) comprises amino acid substitutions at one or more amino acid residues selected from the group consisting of L44, I61, V63, E77, Q82, K83, E84, V93, D95, L96, K98, N100, R107, G109, N110, and V132.

[0197] In the present application, the mutant in the fusion protein may comprise an amino acid substitution at an amino acid residue selected from the following groups: (1) I61, V63, E77, E84, V93, L96, K98, N100, N110, and V132; (2) I61, E77, Q82, K83, E84, and N110; (3) I61, V63, K83, E84, N110, and V132; (4) I61, E77, E84, R107\N110, and V132; (5) I61, V63, E77, K83, E84, N100, and N110; (6) I61, E77, Q82, K83, E84, R107, and N110; (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109, N110, and V132; (8) I61, E77, Q82, K83, E84, V132, and N110; (9) I61 and N110; (10) I61, D95, L96, G109, N110, and V132; (11) I61, D95, L96, K98, G109, N110, and V132; (12) I61, E77, E84, V93, R107, N110, and V132; (13) E77, L96, N100, G109, N110, and V132; (14) I61, V63, Q82, E84, D95, L96, N100, N110, and V132; (15) I61, E77, Q82, K83, E84, V93, D95, L96, K98, N100, N110, and V132; (16) I61, E77, Q82, K83, E84, V93, and N110; (17) I61, V63, E77, K83, E84, D95, L96, K98, N100, and N110; (18) I61, V63, E77, K83, D95, L96, K98, N100, G109, and N110; (19) I61, E77, Q82, E84, V93, D95, L96, K98, N100, and N110; (20) I61, V63, E77, Q82, E84, and N110; and (21) L44V, I61F, E77I, Q82R, K83R, E84Q, N110A, and V132I.

[0198] In the present application, the mutant in the fusion protein may comprise amino acid substitutions selected from the following groups: (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G, N110A, and V132L; (2) I61V, E77N, Q82S, K83R, E84H, and N110A; (3) I61F, V63I, K83R, E84K, N110A, and V132I; (4) I61L, E77Q, E84D, R107N, N110A, and V132I; (5) I61L, V63I, E77K, K83R, E84D, N100G, and N110A; (6) I61V, E77H, Q82R, K83R, E84H, R107S, and N110A; (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R, N110A, and V132R; (8) I61L, E77M, Q82G, K83R, E84D, N110A, and V132L; (9) I61L and N110A; (10) I61F, D95H, L96S, G109H, N110A, and V132S; (11) I61F, D95H, L96S, K98R, G109H, N110A, and V132S; (12) I61L, E77Q, E84D, V93A, R107N, N110A, and V132I; (13) E77K, L96S, N100K, G109H, N110A, and V132L; (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D, N110A, and V132I; (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D, N110A, and V132L; (16) I61V, E77N, Q82S, K83R, E84H, V93A, and N110A; (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R, N100E, and N110A; (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D, G109R, and N110A; (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R, N100G, and N110A; (20) I61L, V63I, E77N, Q82G, E84G, and N110A; and (21) L44V, I61F, E77I, Q82R, K83R, E84Q, N110A, and V132I.

[0199] In the present application, based on the truncated domain of human SIRPα variant 1 (the amino acid sequence shown in SEQ ID NO: 23, i.e., residues 33 - 149 in the amino acid sequence of human SIRPα variant 1), mutants of SIRPα variant 1 respectively containing the amino acid substitution groups of (1) - (21) above can be named M1N, M5N, M12N, M35N, M37N, M41N, M57N, M67N, M81N, M82N, M84N, M91N, M99N, M102N, M111N, M122N, M126N, M130N, M135N, M145N, and M169N in sequence. The mutants of SIRPα variant 1 can contain the amino acid sequences shown in SEQ ID NO: 1 - 21 in sequence. In the present application, the mutants of SIRPα variant 1 can contain amino acid sequences 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 homology with the amino acid sequences shown in any one of SEQ ID NO: 1 - 21.

[0200] First binding domain that specifically binds to CLDN18.2

[0201] In the present application, the first binding domain contains an antigen - binding protein. In the present application, the antigen - binding protein can contain an antibody or an antigen - binding fragment. In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibody, single - chain antibody, chimeric antibody, humanized antibody, and fully human antibody. In certain embodiments, 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.

[0202] In certain embodiments, the antigen - binding protein is capable of binding to CLDN18.2.

[0203] In the present application, the antigen - binding protein can contain at least one CDR of the heavy - chain variable region of an antibody, and the heavy - chain variable region of the antibody can contain the amino acid sequence shown in SEQ ID NO: 24. For example, the CDR can be obtained by partitioning according to the IMGT numbering scheme.

[0204] In certain embodiments, the antigen - binding protein contains HCDR3, and the HCDR3 can contain the amino acid sequence shown in SEQ ID NO: 25.

[0205] In the present application, the antigen - binding protein can contain HCDR2, and the HCDR2 can contain the amino acid sequence shown in SEQ ID NO: 26.

[0206] In the present application, the antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0207] In the present application, the antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3. The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0208] In the present application, the antigen-binding protein may comprise VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0209] In the present application, the antigen-binding protein may comprise an immunoglobulin single variable domain. In the present application, the antigen-binding protein may comprise V H H. For example, the antigen-binding protein may comprise at least one CDR in V H H, and the V H H comprises the amino acid sequence shown in SEQ ID NO: 24. For example, the CDR may be obtained by division according to the IMGT numbering scheme.

[0210] In the present application, the V H H may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0211] In the present application, the V H H may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0212] In the present application, the V H H may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27.

[0213] In the present application, the V H H may comprise HCDR1, HCDR2 and HCDR3. The HCDR1 may comprise the amino acid sequence shown in SEQ IDNO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 25.

[0214] In the present application, the antigen-binding protein may comprise an Fc fragment of an antibody heavy chain constant region. In the present application, the heavy chain constant region of the antigen-binding protein may be derived from IgG. In the present application, the heavy chain constant region may be derived from human IgG. In the present application, the IgG may be selected from IgG1 and IgG4. In the present application, the Fc region of the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 28.

[0215] Linkage between the first binding domain and the second binding domain

[0216] In the present application, the first binding domain may be located at the N-terminus of the second binding domain. For example, the C-terminus of the first binding domain may be directly or indirectly connected to the N-terminus of the second binding domain. For example, the C-terminus of the first binding domain may be indirectly connected to the N-terminus of the second binding domain through a linker. For example, the C-terminus of the first binding domain may also be directly connected to the N-terminus of the second binding domain.

[0217] In the present application, the fusion protein may further comprise 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. For example, in the fusion protein, the C-terminus of the first binding domain may be connected to the N-terminus of the linker, and the C-terminus of the linker may be connected to the N-terminus of the second binding domain. For example, in the fusion protein, from the N-terminus to the C-terminus, it may sequentially comprise the first binding domain, the linker, and the second binding domain.

[0218] In the present application, the linker may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0219] In certain cases, the fusion protein may comprise at least 2 (e.g., 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) of the second binding domains. In the present application, each of the second binding domains may be respectively located at the C-terminus of the first binding domain. In the present application, the two or more second binding domains may be respectively directly or indirectly connected to the C-terminus of the first binding domain.

[0220] In the present application, the fusion protein may comprise a first binding domain that specifically binds to CLDN18.2 and a second binding domain that specifically binds to the CD47 protein, wherein the second binding domain may comprise a mutant of human SIRPα variant 1, and the C-terminus of an antibody or its antigen-binding fragment or variant that specifically binds to CLDN18.2 may be directly or indirectly linked to the N-terminus of the mutant of human SIRPα variant 1. For example, the second binding domain may comprise at least two mutants of human SIRPα variant 1, and the N-termini of the two mutants of human SIRPα variant 1 are respectively linked to the C-terminus of an antibody or its antigen-binding fragment or variant that specifically binds to CLDN18.2.

[0221] In the present application, the fusion protein may comprise at least one polypeptide chain, wherein the polypeptide chain may sequentially comprise, from the N-terminus to the C-terminus, a VH that can specifically bind to CLDN18.2 H an Fc domain, a linker, and a mutant of human SIRPα variant 1. In the present application, the fusion protein may comprise two polypeptide chains, wherein each polypeptide chain may sequentially comprise, from the N-terminus to the C-terminus, a VH that can specifically bind to CLDN18.2 H an Fc domain, a linker, and a mutant of human SIRPα variant 1. For example, the fusion protein may comprise two polypeptide chains, and the two polypeptide chains may comprise the same amino acid sequence, for example, the amino acid sequence shown in any one of SEQ ID NOs: 30-35.

[0222] For example, as Figure 1 shown, the first binding domain of the fusion protein may comprise NA3S-H1, and the second binding domain of the fusion protein may comprise a mutant of human SIRPα variant 1. The first binding domain and the second binding domain may be linked by a linker. The linker may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0223] For example, the polypeptide chain of the fusion protein may sequentially comprise, from the N-terminus to the C-terminus, the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 1, respectively.

[0224] For example, the polypeptide chain of the fusion protein may sequentially comprise, from the N-terminus to the C-terminus, the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 2, respectively.

[0225] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 3 from the N-terminus to the C-terminus.

[0226] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 4 from the N-terminus to the C-terminus.

[0227] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 5 from the N-terminus to the C-terminus.

[0228] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 6 from the N-terminus to the C-terminus.

[0229] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 7 from the N-terminus to the C-terminus.

[0230] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 8 from the N-terminus to the C-terminus.

[0231] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 9 from the N-terminus to the C-terminus.

[0232] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 10 from the N-terminus to the C-terminus.

[0233] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 11 from the N-terminus to the C-terminus.

[0234] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 12 from the N-terminus to the C-terminus.

[0235] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 13 from the N-terminus to the C-terminus.

[0236] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 14 from the N-terminus to the C-terminus.

[0237] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 15 from the N-terminus to the C-terminus.

[0238] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 16 from the N-terminus to the C-terminus.

[0239] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 17 from the N-terminus to the C-terminus.

[0240] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 18 from the N-terminus to the C-terminus.

[0241] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 19 from the N-terminus to the C-terminus.

[0242] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:20 from the N-terminus to the C-terminus.

[0243] For example, the polypeptide chain of the fusion protein may sequentially include the amino acid sequences shown in SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:21 from the N-terminus to the C-terminus.

[0244] Immunoconjugates, nucleic acid molecules, vectors, cells and preparation methods

[0245] On the other hand, the present application also provides an immunoconjugate comprising the fusion protein. For example, the immunoconjugate may be a fusion protein-drug conjugate (ADC), wherein the fusion protein described in the present application is conjugated to one or more therapeutic agents, and the therapeutic agents include but are not limited to cytotoxic agents, radiotoxic agents (e.g., radioisotopes), and / or immunosuppressive agents (e.g., any agent that kills cells by inhibiting immune responses, etc.). In certain embodiments, the therapeutic agent may be a therapeutic agent capable of treating tumor-related diseases or disorders.

[0246] The conjugation may be carried out through a peptide linker (e.g., a cleavable linker) or other means. For example, the linker may be an acid-labile linker, a peptidase-sensitive linker, a photo-labile linker, etc.

[0247] On the other hand, the present application provides one or more isolated nucleic acid molecules encoding the fusion protein or the immunoconjugate. For example, each of the one or more nucleic acid molecules may encode the entire antibody or its antigen-binding fragment, or may encode a part thereof (e.g., one or more of HCDR1-3, LCDR1-3, VL, VH, light chain, or heavy chain).

[0248] The nucleic acid molecule described in the present application may be isolated. For example, it may be produced or synthesized by the following methods: (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) amplification, (ii) produced by recombinant cloning, (iii) purified, e.g., by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthesized, e.g., by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0249] Recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T.J. Silhavy, M.L. Bennan and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F.M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, the nucleic acids can be prepared from genomic DNA fragments, cDNA and RNA, all of which can be directly extracted from cells or recombinantly produced by various amplification methods including, but not limited to, PCR and RT-PCR.

[0250] The direct chemical synthesis of nucleic acids generally involves the sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the 5'-hydroxyl terminus of a growing nucleotide polymer chain, where each addition is achieved by nucleophilic attack of the 5'-hydroxyl terminus of the growing chain on the 3'-position of the monomer being added, which monomer is typically a phosphorus derivative such as a phosphotriester, phosphoramidite, etc. See, e.g., Matteuci et al., Tet. Lett. 521:719 (1980); U.S. Patent No. 4,500,707 to Caruthers et al.; and U.S. Patents Nos. 5,436,327 and 5,700,637 to Southern et al.; On the other hand, the present application provides a vector comprising the isolated polynucleotide of the present application. 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. In some embodiments, the vector is an expression vector, e.g., a phage display vector.

[0251] On the other hand, the present application provides one or more vectors, which contain the nucleic acid molecules described above. For example, the vector may contain one or more nucleic acid molecules described in the present application. Each vector may contain one or more of the nucleic acid molecules. In addition, other genes may also be contained in the vector, such as marker genes that allow the selection of the vector in a suitable host cell and under suitable conditions. In addition, the vector may also contain expression control elements that allow the correct expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' untranslated sequences that are respectively involved in the initiation of transcription and translation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for functionally linking nucleic acids for transcriptional control. The expression control sequence may also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and their artificial hybrid promoters (such as CMV), where a certain part of the promoter may be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, which may or may not contain additional introns. One or more nucleic acid molecules described in the present application may be operably linked to the expression control element.

[0252] The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in genetic engineering, for example. In certain embodiments, the vector may be an expression vector.

[0253] 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. Non-limiting examples of suitable selectable markers for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

[0254] On the other hand, the present application provides a cell comprising the fusion protein, the immunoconjugate, the nucleic acid molecule, or the vector. The cell can be a host cell. For example, the cell can include many cell types such as prokaryotic cells like Escherichia coli or Bacillus subtilis, fungal cells like yeast cells or Aspergillus, insect cells like Drosophila S2 cells or Sf9, or animal cells like fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0255] For example, the vector can be stably or transiently introduced into the host cell by a variety of established techniques. For example, one method involves calcium chloride treatment, where the vector is introduced by calcium precipitation. Other salts can be used in a similar manner, such as calcium phosphate. Additionally, electroporation (i.e., applying an electric current to increase the permeability of the cell to nucleic acids) can be used. Other examples of transformation methods include microinjection, DEAE-dextran-mediated transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes, and dendrimers can also be used to transfect host cells.

[0256] When introducing a heterologous sequence into a host cell, a variety of methods can be implemented to identify the host cells into which the vector has been introduced. An exemplary selection method involves subculturing individual cells to form single colonies and then testing for the expression of the desired protein product. Another method requires selecting host cells containing the heterologous sequence based on phenotypic traits conferred by the expression of a selectable marker gene contained within the vector.

[0257] For example, the introduction of various heterologous sequences of the present application into host cells can be confirmed by methods such as PCR, Southern blot, or Northern blot hybridization. For example, nucleic acids can be prepared from the resulting host cells, and specific target sequences can be amplified by PCR using primers specific to the target sequence. The amplification products are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis and then stained with ethidium bromide, SYBR Green solution, etc., or detected by UV to detect DNA. Alternatively, nucleic acid probes specific to the target sequence can be used in a hybridization reaction. The expression of a specific gene sequence can be determined by reverse transcription corresponding to the mRNA by PCR, Northern blot hybridization, or by immunodetection using an antibody that reacts with the encoded gene product. Exemplary immunoassays include, but are not limited to, ELISA, radioimmunoassay, and sandwich immunoassay.

[0258] In addition, the introduction of various heterologous sequences of the present application into host cells can be confirmed by the enzymatic activity of the enzymes encoded by the heterologous sequences (e.g., enzymatic markers). Enzymes can be assayed by a variety of methods known in the art. Generally, enzymatic activity can be determined by the formation of a product or the conversion of a substrate of the enzymatic reaction under study. The reaction can be carried out in vitro or in vivo.

[0259] On the other hand, the present application provides a method for preparing the fusion protein described above, which may include culturing the cells under conditions that enable the expression of the fusion protein. For example, it can be achieved by using an appropriate culture medium, appropriate temperature, culture time, etc., which are known to those of ordinary skill in the art.

[0260] In certain cases, the method may further include the steps of isolating and / or purifying the fusion protein. For example, affinity chromatography can be performed using protein G-agarose or protein A-agarose, and the fusion protein described in the present application can also be purified and isolated by gel electrophoresis and / or high performance liquid chromatography, etc.

[0261] Compositions and applications

[0262] On the other hand, the present application provides a composition comprising the fusion protein, the immunoconjugate, or the nucleic acid molecule described above, and optionally a pharmaceutically acceptable excipient.

[0263] For example, the pharmaceutically acceptable excipient 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.

[0264] In the present application, the 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, operating according to the techniques disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0265] In the present application, 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, or administration through a subcutaneous depot.

[0266] For example, the composition can be used to inhibit tumor growth. For example, the composition of the present application can inhibit or delay the development or progression of a disease, can reduce the tumor size (even substantially eliminate the tumor), and / or can alleviate and / or stabilize the disease state.

[0267] For example, the composition described in the present 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 sterile solutions, suspensions or emulsions, or for topical administration as an ointment or cream or for rectal administration as a suppository. The composition can be in unit dosage form suitable for single administration of a precise dose. The composition can further comprise conventional pharmaceutical carriers or excipients. In addition, the composition can include other drugs or medicaments, carriers, adjuvants, etc.

[0268] The composition described in the present application can comprise a therapeutically effective amount of the fusion protein. The therapeutically effective amount is the dose required to prevent and / or treat (at least in part) a disorder or condition (such as a tumor) and / or any complications thereof in a subject suffering from or at risk of developing the same. The specific amount / concentration of the dose can vary according to the administration method and the needs of the patient, and can be determined based on, for example, the patient's volume, viscosity and / or body weight, etc. It should be understood that those skilled in the art (such as doctors or pharmacists) can conveniently adjust these specific doses based on the condition of a particular patient, formulation and / or disease.

[0269] In the present application, the terms "treat" or "cure" or "alleviate" or "improve" are used interchangeably in the present application and refer to a method of obtaining a beneficial or desired result (including but not limited to a therapeutic benefit and / or a prophylactic benefit). In the present application, a therapeutic benefit generally refers to eradicating or alleviating the severity of the underlying disorder being treated. In addition, a therapeutic benefit is achieved by eradicating, alleviating the severity or reducing the incidence of one or more physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder. For a prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease or to a subject reporting one or more physiological symptoms of the disease, even if the disease may not have been diagnosed.

[0270] On the other hand, the present application provides the use of the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the composition, or the cell described above in the preparation of a medicament, wherein the medicament can be used to prevent and / or treat a disease and / or a disorder.

[0271] On the other hand, the present application provides the fusion protein, immunoconjugate, nucleic acid molecule, vector, composition or cell described above for preventing and / or treating a disease and / or a disorder.

[0272] On the other hand, the present application provides a method for preventing and / or treating a disease and / or disorder, the method comprising administering to a subject the fusion protein, immunoconjugate, nucleic acid molecule, vector, composition or cell described in the present application.

[0273] On the other hand, the present application provides a method for inhibiting the growth and / or proliferation of a tumor or tumor cells, the method may comprise contacting the fusion protein or composition described in the present application with the tumor or tumor cells. For example, the contacting may occur in vitro.

[0274] On the other hand, the present application provides a method for inhibiting the growth and / or proliferation of a tumor or tumor cells, which may comprise administering an effective amount of the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the composition, or the cell to a subject in need thereof.

[0275] In the present application, the disease and / or disorder may include a disease and / or disorder associated with CLDN18.2.

[0276] In the present application, the disease and / or disorder associated with CLDN18.2 includes a disease involving cells expressing CLDN18.2 or a disease associated with cells expressing CLDN18.2.

[0277] In the present application, the tumor may include solid tumors and non-solid tumors.

[0278] In the present application, the tumor may include gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and / or gallbladder cancer.

[0279] On the other hand, the present application provides the fusion protein, the immunoconjugate, the nucleic acid molecule, the vector, the composition, or the cell, which can prevent and / or treat a disease and / or disorder.

[0280] In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, goats, rabbits, mice, rats, or monkeys.

[0281] The present application also includes the following technical solutions:

[0282] 1. A fusion protein, comprising:

[0283] A first binding domain that specifically binds to CLDN18.2; and

[0284] A second binding domain that specifically binds to the CD47 protein;

[0285] Among them, the second binding domain has at least one of the following characteristics:

[0286] (a) binds to CD47 protein with a KD value of 4×10-8 M or lower;

[0287] (b) specifically blocks the interaction between CD47 protein and SIRPα;

[0288] (c) does not cause a coagulation reaction; and

[0289] (d) can inhibit the growth and / or proliferation of tumors or tumor cells.

[0290] 2. The fusion protein according to embodiment 1, wherein the second binding domain comprises a mutant of human SIRPα variant 1, and the mutant comprises amino acid residue mutations at one or more positions compared with the amino acid sequence from position 33 to position 149 of SEQ ID NO: 22.

[0291] 3. The fusion protein according to embodiment 2, wherein the mutant comprises amino acid mutations at one or more amino acid positions selected from the group consisting of: L44, I61, V63, E77, Q82, K83, E84, V93, D95, L96, K98, N100, R107, G109 and V132.

[0292] 4. The fusion protein according to any one of embodiments 2-3, wherein the mutant comprises amino acid substitutions at the I61 and E77 amino acid residues.

[0293] 5. The fusion protein according to any one of embodiments 2-4, wherein the mutant comprises amino acid substitutions at the I61 and V132 amino acid residues.

[0294] 6. The fusion protein according to any one of embodiments 2-5, wherein the mutant comprises amino acid substitutions at the I61 and E84 amino acid residues.

[0295] 7. The fusion protein according to any one of embodiments 2-6, wherein the mutant comprises amino acid substitutions at the I61, K83 and E84 amino acid residues.

[0296] 8. The fusion protein according to any one of embodiments 2-7, wherein the mutant comprises amino acid substitutions at the I61, G109 and V132 amino acid residues.

[0297] 9. The fusion protein according to any one of embodiments 2-8, wherein the mutant comprises amino acid substitutions at the I61, D95, L96, G109 and V132 amino acid residues.

[0298] 10. The fusion protein according to any one of embodiments 2-9, wherein the mutant comprises amino acid substitutions at amino acid residues V93 and R107.

[0299] 11. The fusion protein according to any one of embodiments 2-10, wherein the mutant comprises amino acid substitutions at amino acid residues L44 and I61.

[0300] 12. The fusion protein according to any one of embodiments 2-11, wherein the mutant comprises amino acid substitutions at amino acid residues L44 and E77.

[0301] 13. The fusion protein according to any one of embodiments 2-12, wherein the mutant comprises amino acid substitutions at amino acid residues Q82, K83 and E84.

[0302] 14. The fusion protein according to any one of embodiments 2-13, wherein the mutant comprises amino acid substitutions at amino acid residues E77 and V132.

[0303] 15. The fusion protein according to any one of embodiments 2-14, wherein the mutant comprises amino acid mutations at amino acid residues selected from the group consisting of:

[0304] (1) I61, V63, E77, E84, V93, L96, K98, N100 and V132;

[0305] (2) I61, E77, Q82, K83 and E84;

[0306] (3) I61, V63, K83, E84 and V132;

[0307] (4) I61, E77, E84, R107 and V132;

[0308] (5) I61, V63, E77, K83, E84 and N100;

[0309] (6) I61, E77, Q82, K83, E84 and R107;

[0310] (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109 and V132;

[0311] (8) I61, E77, Q82, K83, E84 and V132;

[0312] (9) I61;

[0313] (10) I61, D95, L96, G109 and V132;

[0314] (11) I61, D95, L96, K98, G109 and V132;

[0315] (12) I61, E77, E84, V93, R107 and V132;

[0316] (13) E77, L96, N100, G109 and V132;

[0317] (14) I61, V63, Q82, E84, D95, L96, N100 and V132;

[0318] (15) I61, E77, Q82, K83, E84, V93, D95, L96, K98, N100 and V132;

[0319] (16) I61, E77, Q82, K83, E84 and V93;

[0320] (17) I61, V63, E77, K83, E84, D95, L96, K98 and N100;

[0321] (18) I61, V63, E77, K83, D95, L96, K98, N100 and G109;

[0322] (19) I61, E77, Q82, E84, V93, D95, L96, K98 and N100;

[0323] (20) I61, V63, E77, Q82 and E84; and

[0324] (21) L44, I61, E77, Q82, K83, E84 and V132.

[0325] 16. The fusion protein according to any one of embodiments 2 - 15, wherein the mutant comprises one or more amino acid substitutions selected from the group consisting of: L44V, I61L / V / F, V63I, E77I / N / Q / K / H / M / R / V / L, Q82S / R / G / N, K83R, E84Q / K / 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.

[0326] 17. The fusion protein according to any one of embodiments 2 - 16, wherein the mutant comprises the amino acid substitutions I61L / V / F and E77Q / N / I.

[0327] 18. The fusion protein according to any one of embodiments 2-17, wherein the mutant comprises amino acid substitutions of I61L / V / F and V132I / S.

[0328] 19. The fusion protein according to any one of embodiments 2-18, wherein the mutant comprises amino acid substitutions of I61L / V / F, K83R and E84Q / D / H.

[0329] 20. The fusion protein according to any one of embodiments 2-19, wherein the mutant comprises amino acid substitutions of I61L / V / F, G109H and V132I / S.

[0330] 21. The fusion protein according to any one of embodiments 2-20, wherein the mutant comprises amino acid substitutions of I61L / V / F, D95H, L96S, G109H and V132I / S.

[0331] 22. The fusion protein according to any one of embodiments 2-21, wherein the mutant comprises amino acid substitutions of V93A and R107N.

[0332] 23. The fusion protein according to any one of embodiments 2-22, wherein the mutant comprises amino acid substitutions of L44V and I61L / V / F.

[0333] 24. The fusion protein according to any one of embodiments 2-23, wherein the mutant comprises amino acid substitutions of L44V and E77Q / N / I.

[0334] 25. The fusion protein according to any one of embodiments 2-24, wherein the mutant comprises amino acid substitutions of Q82S / R / G / N, K83R and E84Q / K / H / D / R / G.

[0335] 26. The fusion protein according to any one of embodiments 2-25, wherein the mutant comprises amino acid substitutions of E77Q / N / I and V132I / S.

[0336] 27. The fusion protein according to any one of embodiments 2-26, wherein the mutant comprises amino acid mutations selected from the group consisting of:

[0337] (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G and V132L;

[0338] (2) I61V, E77N, Q82S, K83R and E84H;

[0339] (3) I61F, V63I, K83R, E84K, and V132I;

[0340] (4) I61L, E77Q, E84D, R107N, and V132I;

[0341] (5) I61L, V63I, E77K, K83R, E84D, and N100G;

[0342] (6) I61V, E77H, Q82R, K83R, E84H, and R107S;

[0343] (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R, and V132R;

[0344] (8) I61L, E77M, Q82G, K83R, E84D, and V132L;

[0345] (9) I61L;

[0346] (10) I61F, D95H, L96S, G109H, and V132S;

[0347] (11) I61F, D95H, L96S, K98R, G109H, and V132S;

[0348] (12) I61L, E77Q, E84D, V93A, R107N, and V132I;

[0349] (13) E77K, L96S, N100K, G109H, and V132L;

[0350] (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D, and V132I;

[0351] (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D, and V132L;

[0352] (16) I61V, E77N, Q82S, K83R, E84H, and V93A;

[0353] (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R, and N100E;

[0354] (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D, and G109R;

[0355] (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R, and N100G;

[0356] (20) I61L, V63I, E77N, Q82G, and E84G and

[0357] (21) L44V, I61F, E77I, Q82R, K83R, E84Q, and V132I.

[0358] 28. The fusion protein according to any one of embodiments 2-27, wherein the mutant further comprises modification of one or more glycosylation sites.

[0359] 29. The fusion protein according to embodiment 28, wherein the modification of the glycosylation site comprises a glycosylation site mutation.

[0360] 30. The fusion protein according to embodiment 29, wherein the glycosylation site mutation is located at position N110.

[0361] 31. The fusion protein according to any one of embodiments 29-30, wherein the glycosylation site mutation comprises an amino acid substitution of N110A.

[0362] 32. The fusion protein according to any one of embodiments 2-31, wherein the mutant comprises the amino acid sequence shown in any one of SEQ ID NO: 1-21.

[0363] 33. The fusion protein according to any one of embodiments 1-33, wherein the first binding domain comprises an antigen-binding protein targeting CLDN18.2.

[0364] 34. The fusion protein according to embodiment 33, wherein the antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0365] 35. The fusion protein according to embodiment 34, wherein the antibody is selected from the group consisting of: monoclonal antibody, single-chain antibody, chimeric antibody, humanized antibody, and fully human antibody.

[0366] 36. The fusion protein according to any one of embodiments 34-35, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab’, F(ab)2, dAb, isolated complementarity-determining region CDR, Fv, and scFv.

[0367] 37. The fusion protein according to any one of embodiments 1-36, wherein the CLDN18.2 comprises human CLDN18.2.

[0368] 38. The fusion protein according to any one of embodiments 1-37, wherein the first binding domain comprises at least one CDR in the heavy chain variable region (VH), and the VH comprises the amino acid sequence shown in SEQ ID NO: 24.

[0369] 39. The fusion protein according to any one of embodiments 1-38, wherein the first binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0370] 40. The fusion protein according to any one of embodiments 1-39, wherein the first binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26.

[0371] 41. The fusion protein according to any one of embodiments 1-40, wherein the first binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0372] 42. The fusion protein according to any one of embodiments 1-41, wherein the first binding domain comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 24.

[0373] 43. The fusion protein according to any one of embodiments 1-42, wherein the first binding domain comprises an immunoglobulin single variable domain.

[0374] 44. The fusion protein according to embodiment 43, wherein the immunoglobulin single variable domain comprises VHH.

[0375] 45. The fusion protein according to any one of embodiments 1-44, wherein the first binding domain comprises at least one CDR in VHH, and the VHH comprises the amino acid sequence shown in SEQ ID NO: 24.

[0376] 46. The fusion protein according to any one of embodiments 44-45, wherein the VHH comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 25.

[0377] 47. The fusion protein according to any one of embodiments 44-46, wherein the VHH comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26.

[0378] 48. The fusion protein according to any one of embodiments 44 - 47, wherein the VHH comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0379] 49. The fusion protein according to any one of embodiments 44 - 48, wherein the VHH comprises the amino acid sequence shown in SEQ ID NO: 24.

[0380] 50. The fusion protein according to any one of embodiments 1 - 49, wherein the first binding domain comprises a heavy chain constant region Fc fragment.

[0381] 51. The fusion protein according to embodiment 50, wherein the heavy chain constant region is derived from IgG.

[0382] 52. The fusion protein according to any one of embodiments 50 - 51, wherein the heavy chain constant region is derived from human IgG.

[0383] 53. The fusion protein according to any one of embodiments 51 - 52, wherein the IgG is selected from the group consisting of: IgG1 and IgG4.

[0384] 54. The fusion protein according to any one of embodiments 50 - 53, wherein the heavy chain constant region Fc fragment comprises the amino acid sequence shown in SEQ ID NO: 28.

[0385] 55. The fusion protein according to any one of embodiments 1 - 55, wherein the first binding domain is located at the N - terminus of the second binding domain.

[0386] 56. The fusion protein according to any one of embodiments 1 - 55, wherein the fusion protein further comprises a linker, and the linker is located at the C - terminus of the first binding domain and at the N - terminus of the second binding domain.

[0387] 57. The fusion protein according to embodiment 56, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 29.

[0388] 58. The fusion protein according to any one of embodiments 1 - 57, which comprises at least 2 of the second binding domains.

[0389] 59. The fusion protein according to embodiment 58, wherein each of the second binding domains is located at the C - terminus of the first binding domain, respectively.

[0390] 60. The fusion protein according to any one of embodiments 1 - 59, which comprises at least one polypeptide chain, and the polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 30 - 35.

[0391] 61. The fusion protein according to any one of embodiments 1-60, which comprises two polypeptide chains, and the two polypeptide chains comprise the same amino acid sequence.

[0392] 62. An immunoconjugate, which comprises the fusion protein according to any one of embodiments 1-61.

[0393] 63. One or more isolated nucleic acid molecules, which encode the fusion protein according to any one of embodiments 1-61 or the immunoconjugate according to embodiment 62.

[0394] 64. A vector, which comprises the nucleic acid molecule according to embodiment 63.

[0395] 65. A pharmaceutical composition, which comprises the fusion protein according to any one of embodiments 1-61, the immunoconjugate according to embodiment 62, the nucleic acid molecule according to embodiment 63, and optionally a pharmaceutically acceptable carrier.

[0396] 66. A cell, which comprises the fusion protein according to any one of embodiments 1-61, the immunoconjugate according to embodiment 62, the nucleic acid molecule according to embodiment 63, or the vector according to embodiment 64.

[0397] 67. A method for preparing the fusion protein according to any one of embodiments 1-61, which comprises culturing the cell according to embodiment 66 under conditions that enable the expression of the fusion protein.

[0398] 68. Use of the fusion protein according to any one of embodiments 1-61, the immunoconjugate according to embodiment 62, the nucleic acid molecule according to embodiment 63, the vector according to embodiment 64, the pharmaceutical composition according to embodiment 65, or the cell according to embodiment 66 in the preparation of a medicament for preventing and / or treating a disease and / or disorder.

[0399] 69. The use according to embodiment 68, wherein the disease and / or disorder comprises a disease and / or disorder related to CLDN18.2.

[0400] 70. The use according to any one of embodiments 68-69, wherein the disease and / or disorder related to CLDN18.2 includes a disease involving cells expressing CLDN18.2 or a disease related to cells expressing CLDN18.2.

[0401] 71. The use according to any one of embodiments 68-70, wherein the disease and / or disorder includes a tumor.

[0402] 72. The use according to embodiment 71, wherein the tumor includes solid tumors and / or non-solid tumors.

[0403] 73. The use according to any one of embodiments 71-72, wherein the tumor includes gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and / or gallbladder cancer.

[0404] Without being limited by any theory, the following examples are merely for illustrating the various technical solutions of the present invention and are not used to limit the scope of the present invention.

[0405] Examples

[0406] Example 1 Construction of fusion protein

[0407] With reference to the fusion protein structure as Figure 1 shown, taking CS001 as an example, from the N-terminus to the C-terminus, linker 1 (amino acid sequence as shown in SEQ ID NO: 29) was selected and sequentially linked to Claudin18.2 nanobody NA3S-H1, linker, and SIRPα mutant M91N (amino acid sequence as shown in SEQ ID NO: 12), wherein the N-terminus of M91N and the C-terminus of the heavy chain of NA3S-H1 were linked by a linker to obtain the fusion protein CS001.

[0408] For CS002, CS003, CS004, CS005, and CS006, M91N was replaced with the corresponding SIRPα mutants 002N, M5N, M169N, M82N, and M84N according to Table 1 on the basis of CS001. Among them, 002N was obtained by mutating the amino acid sequence shown in SEQ ID NO: 23 at N110A.

[0409] The amino acid sequences of HCDR1-3 of the nanobody NA3S-H1 are shown in SEQ ID NO: 27, SEQ ID NO: 26, and SEQ ID NO: 25 respectively, and the amino acid sequence of VHH is shown in SEQ ID NO: 24. The amino acid sequence of IgG Fc is shown in SEQ ID NO: 28.

[0410] Table 1 Composition of each fusion protein

[0411]

[0412]

[0413] Example 2 Detection of the activity of binding to dual antigens

[0414] (1) ELISA was used to evaluate the binding activity of the bifunctional fusion protein and CD47.

[0415] Coat ELISA strips with CD47 (CD47 Protein, Human, Recombinant (ECD, His Tag), Sino Biological) and incubate overnight at 4°C; after washing with PBST, add 10% fetal bovine serum and block at 37°C for 1 hour; add different concentrations of bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005 and CS006 and react at 37°C for 1 hour; after washing with PBST, add goat anti-human IgG Fc secondary antibody conjugated with horseradish peroxidase (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) and react at 37°C for 30 minutes; wash 5 times with PBST; add 100 μL of TMB (eBioscience) to each well and incubate in the dark at room temperature (20 ± 5°C) for 1 - 2 min; then add 100 μL of 2N H2SO4 stop solution to each well to terminate the substrate reaction, and read the OD value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader to analyze the binding ability of the bifunctional fusion protein to CD47.

[0416] The results are as Figure 2 shown. Figure 2 It shows that the bifunctional fusion protein CS001 has the strongest binding ability to CD47, and the binding abilities of CS002 and CS003 to CD47 are similar and the weakest.

[0417] (2) Evaluate the binding activity of the bifunctional fusion protein and the CD47-positive cell line Raji cells by flow cytometry.

[0418] Collect human lymphoma tumor cells Raji and add them to 1.5 mL EP tubes at 5 × 10 5 cells per tube; add different concentrations of bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005 and CS006 and incubate on ice in the dark for 30 min; after washing with FACS wash buffer, add goat anti-human IgG Fc secondary antibody labeled with PE (Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen) and incubate on ice in the dark for 30 min; wash 2 times with FACS wash buffer; add 400 μL of 1% paraformaldehyde fixative (Solarbio) to each tube to fix the cells, mix well and then detect the relative fluorescence intensity and positive rate of PE fluorescence by flow cytometry to analyze the binding ability of the bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005 and CS006 to Raji cells.

[0419] The results are as Figures 3A - 3B shown. Figures 3A - 3B It shows that the bifunctional fusion protein CS001 has the strongest binding ability to CD47-positive tumor cells Raji, and CS002 has the weakest binding ability to Raji cells.

[0420] (3) Using the Claudin18.2 nanobody NA3S-H1 as a control, the binding activity of the bifunctional fusion protein and HEK-293T cells overexpressing the human Claudin18.2 gene (abbreviated as 293T / CLDN18.2) was evaluated by flow cytometry.

[0421] Collect 293T / CLDN18.2 cells and add them to 1.5 mL EP tubes at a density of 5×10 5 cells per tube; add different concentrations of bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005 and CS006, as well as the Claudin18.2 nanobody NA3S-H1, and incubate in the dark on ice for 30 min; after washing with FACS wash buffer, add a PE-fluorescently labeled goat anti-human IgG Fc secondary antibody (Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen), and incubate in the dark on ice for 30 min; wash twice with FACS wash buffer; add 400 μL of 1% paraformaldehyde fixative (Solarbio) to each tube to fix the cells, mix well and then detect the relative fluorescence intensity and positive rate of PE fluorescence by flow cytometry to analyze the binding ability of bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005 and CS006 to 293T / CLDN18.2 cells.

[0422] The results are as Figure 4 shown. Figure 4 It shows that the binding abilities of all bifunctional fusion proteins to 293T / CLDN18.2 cells are similar, the same as the nanobody NA3S-H1.

[0423] Example 3 Activity analysis of blocking the CD47 / SIRPα interaction

[0424] ELISA was used to evaluate the biological activity of the bifunctional fusion protein in blocking the CD47 / SIRPα interaction.

[0425] Coat an ELISA plate with SIRPα (Recombinant Human SIRPA, Sino Biological) at 1 μg / ml and incubate overnight at 4°C. After washing with PBST, add 10% fetal bovine serum and block at 37°C for 1 hour. Add different concentrations of bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005, and CS006 respectively and react at 37°C for 1 hour. After washing with PBST, add biotin-labeled Biotin-CD47 (Recombinant Human CD47-biotin, Jiaxuan Biotechnology) to a final concentration of 2 μg / ml and react at 37°C for 30 min. Wash 5 times with PBST. Add streptavidin-horseradish peroxidase (Streptavidin-HRP, Jiaxuan Biotechnology) and incubate at 37°C for 30 minutes. Wash 5 times with PBST. Add 100 μL of TMB (eBioscience) to each well and place in the dark at room temperature (20 ± 5°C) for 1 - 5 min. Add 100 μL of 2N H2SO4 stop solution to each well to terminate the substrate reaction. Read the OD value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and analyze the blocking effect of bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005, and CS006 on CD47 / SIRPα.

[0426] It can be seen from Figure 5 that the bifunctional fusion protein CS001 has the strongest ability to competitively block the binding of CD47 to SIRPα, and CS002 has the weakest blocking ability.

[0427] Example 4 Analysis of ADCC Activity

[0428] Using the Claudin18.2 nanobody NA3S-H1 as a control, the Claudin18.2-positive cell line 293T / CLDN18.2 as target cells, and Jurkat cells overexpressing the human FcγRIIIa gene and NFAT fluorescent reporter gene (abbreviated as Jurkat-ADCC cells) as effector cells, evaluate the ADCC activity of the bifunctional fusion protein by the reporter gene method.

[0429] Collect 293T / CLDN18.2 cells and add them to a 96-well cell culture plate at 2 × 10 4 cells per tube; collect Jurkat-ADCC cells and add them to each tube at 1.2 × 10 5Cells were added to a 96-well cell culture plate; different concentrations of the functional fusion proteins CS001, CS002, CS003, CS004, CS005, and CS006, as well as the Claudin18.2 nanobody NA3S-H1, were added, and the mixture was incubated in an incubator at 37 °C for 6 hours; 100 μL of luciferase detection solution was added to each well, and after reacting in the dark at room temperature for 10 min, the relative fluorescence intensity value (RLU) of chemiluminescence was read using a microplate reader to analyze the ADCC activity of the bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005, and CS006.

[0430] The results are as Figure 6 shown. Figure 6 It shows that all bifunctional fusion proteins have similar ADCC activities, and the activities are the same as those of the nanobody NA3S-H1.

[0431] Example 5 Analysis of the Activity of Binding to Red Blood Cells

[0432] Flow cytometry was used to evaluate the binding activity of the bifunctional fusion proteins to healthy human red blood cells.

[0433] Healthy human red blood cells were collected and added to 1.5 mL EP tubes at a rate of 1×10 6 cells per tube; different concentrations of the bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005, and CS006 were added, and the mixture was incubated on ice in the dark for 30 min; after washing with FACS wash buffer, PE-fluorescently labeled goat anti-human IgG Fc secondary antibody (Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen) was added, and the mixture was incubated on ice in the dark for 30 min; washed twice with FACS wash buffer; 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells, and after mixing, the relative fluorescence intensity and positive rate of PE fluorescence were detected by flow cytometry to analyze the ability of the bifunctional fusion proteins CS001, CS002, CS003, CS004, CS005, and CS006 to bind to human red blood cells.

[0434] The results are as Figures 7A - 7B shown. Figures 7A - 7B It shows that the bifunctional fusion proteins can all bind to red blood cells to varying degrees. CS001 has the strongest ability to bind to red blood cells, and CS003 has the weakest ability to bind to red blood cells.

[0435] Example 6 In Vivo Tumor Inhibitory Activity of Bifunctional Fusion Proteins

[0436] An SCID mouse model xenografted with HEK-293T cells overexpressing the Claudin18.2 gene (293T / CLDN18.2) was used to evaluate the antitumor activity inhibitory effect of the bifunctional fusion protein.

[0437] 293T / CLDN18.2 cells were inoculated subcutaneously into the right shoulder of female SCID mice, with a total of 24 mice inoculated. When the tumors grew to about 100 mm 3 in size, they were grouped for drug administration. There were a total of 8 groups, with 3 mice in each group, namely the Vehicle group, the IMAB362 (20 mg / kg) group, the NA3S-H1 (10 mg / kg) group, the M91-Fc (10 mg / kg) group, the NA3S-H1 + M91-Fc (10 + 10 mg / kg) group, the CS001 (15 mg / kg) group, the CS002 (15 mg / kg) group, and the CS004 (15 mg / kg) group. They were administered by tail vein injection twice a week for three weeks. The tumor volume and body weight were measured weekly, and the relationship between the body weight and tumor volume changes of the tumor-bearing mice and the drug administration time was recorded. At the end of the experiment, the tumor-bearing mice were euthanized, and the tumors were dissected, weighed, and photographed. The tumor growth inhibition rate TGITV (%) was calculated and statistically analyzed.

[0438] The results were as Figure 8 shown, Figure 8 showing that the tumor growth inhibition rates of the IMAB362, NA3S-H1, M91-Fc, NA3S-H1 + M91-Fc, CS001, CS002, and CS004 groups were 40%, 54%, 36%, 53%, 34%, 96%, and 97% respectively. The tumor volumes of each treatment group were significantly lower than those of the blank control group (p < 0.05), showing a significant antitumor effect and effectively inhibiting tumor growth. The tumor volumes of the CS002 and CS004 groups were similar and the smallest among all treatment groups, significantly lower than those of the M91-Fc (p < 0.01), NA3S-H1 (p < 0.01), and NA3S-H1 + M91-Fc groups (p < 0.05), indicating that their inhibitory effect on tumor activity was superior to that of the single-drug groups and the combined drug group. During the treatment period, the tumor-bearing mice showed good tolerance to the test substances IMAB362, NA3S-H1, M91-Fc, NA3S-H1 + M91-Fc, CS001, CS002, and CS004. The body weights of the mice in each group were normal, without abnormal manifestations, and their general conditions were good.

[0439] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those of ordinary skill in the art and are within the scope of the appended claims and their equivalents. Sequence Listing <110> Hangzhou Shangjian Biotechnology Co., Ltd., Shangjian Monoclonal Antibody (Beijing) Biotechnology Co., Ltd. <120> Fusion Protein Containing SIRPα Mutant <130> 0070-PA-023 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M1N <400> 1 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Ile Leu Ile Tyr 35 40 45 Asn Gln Lys Lys Gly His Phe Pro Arg Val Thr Thr Leu Ser Asp Ser 50 55 60 Thr Arg Arg Gly Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M5N <400> 2 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Val Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Asn Leu Ile Tyr 35 40 45 Asn Ser Arg His Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 3 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M12N <400> 3 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Phe Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gln Arg Lys Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M35N <400> 4 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Gln Leu Ile Tyr 35 40 45 Asn Gln Lys Asp Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Asn Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M37N <400> 5 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Lys Leu Ile Tyr 35 40 45 Asn Gln Arg Asp Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Gly Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 6 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M41N <400> 6 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Val Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg His Leu Ile Tyr 35 40 45 Asn Arg Arg His Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Ser Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 7 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M57N <400> 7 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Ile Leu Ile Tyr 35 40 45 Asn Gly Lys Arg Gly His Phe Pro Arg Val Thr Thr Leu Ser Asp Thr 50 55 60 Thr Lys Arg Gly Asn Met Asp Phe Ser Ile Ser Ile Arg Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Arg Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M67N <400> 8 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Met Leu Ile Tyr 35 40 45 Asn Gly Arg Asp Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M81N <400> 9 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M82N <400> 10 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Phe Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser His Ser 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile His Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ser Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 11 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M84N <400> 11 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Phe Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser His Ser 50 55 60 Thr Arg Arg Asn Asn Met Asp Phe Ser Ile Arg Ile His Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ser Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M91N <400> 12 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Gln Leu Ile Tyr 35 40 45 Asn Gln Lys Asp Gly His Phe Pro Arg Val Thr Thr Ala Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Asn Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 13 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M99N <400> 13 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Lys Leu Ile Tyr 35 40 45 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Ser 50 55 60 Thr Lys Arg Lys Asn Met Asp Phe Ser Ile Arg Ile His Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M102N <400> 14 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gly Lys Gly Gly His Phe Pro Arg Val Thr Thr Val Ser Arg Ser 50 55 60 Thr Lys Arg Asp Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 15 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M111N <400> 15 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Arg Leu Ile Tyr 35 40 45 Asn Asn Arg Gly Gly His Phe Pro Arg Val Thr Thr Leu Ser Glu Thr 50 55 60 Thr Arg Arg Asp Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 16 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M122N <400> 16 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Val Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Asn Leu Ile Tyr 35 40 45 Asn Ser Arg His Gly His Phe Pro Arg Val Thr Thr Ala Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 17 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M126N <400> 17 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Val Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu Ile Tyr 35 40 45 Asn Gln Arg Asp Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Thr 50 55 60 Thr Arg Arg Glu Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 18 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M130N <400> 18 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Val Leu Ile Tyr 35 40 45 Asn Gln Arg Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser 50 55 60 Thr Arg Arg Asp Asn Met Asp Phe Ser Ile Arg Ile Arg Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 19 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M135N <400> 19 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Val Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Leu Leu Ile Tyr 35 40 45 Asn Gly Lys Gly Gly His Phe Pro Arg Val Thr Thr Leu Ser Glu Thr 50 55 60 Thr Arg Arg Gly Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 20 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M145N <400> 20 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Ile Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Asn Leu Ile Tyr 35 40 45 Asn Gly Lys Gly Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 21 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> M169N <400> 21 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Val Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Phe Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Ile Leu Ile Tyr 35 40 45 Asn Arg Arg Gln Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 22 <211> 504 <212> PRT <213> Artificial Sequence <220> <223> Human SIRPa Variant 1 <400> 22 Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys 1 5 10 15 Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu 20 25 30 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 35 40 45 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly 50 55 60 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 65 70 75 80 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 85 90 95 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 100 105 110 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 130 135 140 Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala 145 150 155 160 Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser 165 170 175 Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser 180 185 190 Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser 195 200 205 Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser 210 215 220 Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu 225 230 235 240 Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 245 250 255 Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr 260 265 270 Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu 275 280 285 Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu 290 295 300 Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val 305 310 315 320 Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp 325 330 335 Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His 340 345 350 Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn 355 360 365 Glu Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val 370 375 380 Ala Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys 385 390 395 400 Ala Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn 405 410 415 Ala Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu 420 425 430 Asn Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn 435 440 445 Asn His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser 450 455 460 Glu Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg 465 470 475 480 Thr Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr 485 490 495 Ala Ser Val Gln Val Pro Arg Lys 500 <210> 23 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Human wild-type SIRPα truncated domain (amino acids 33 - 149) <400> 23 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser 115 <210> 24 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> CLDN18.2VHH <400> 24 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 25 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 (IMGT) <400> 25 Asn Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val 1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 (IMGT) <400> 26 Ile Ser Thr Gly Gly Thr Thr 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 (IMGT) <400> 27 Gly Ser Ile Phe Asn Ile Pro Val 1 5 <210> 28 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> IgG1-Fc <400> 28 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 29 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 29 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 30 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> CS001 <400> 30 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile Gln 355 360 365 Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg 370 375 380 Cys Thr Ala Thr Ser Leu Leu Pro Val Gly Pro Ile Gln Trp Phe Arg 385 390 395 400 Gly Ala Gly Pro Gly Arg Gln Leu Ile Tyr Asn Gln Lys Asp Gly His 405 410 415 Phe Pro Arg Val Thr Thr Ala Ser Asp Leu Thr Lys Arg Asn Asn Met 420 425 430 Asp Phe Ser Ile Asn Ile Gly Ala Ile Thr Pro Ala Asp Ala Gly Thr 435 440 445 Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ile Glu Phe 450 455 460 Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser 465 470 475 <210> 31 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> CS002 <400> 31 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile Gln 355 360 365 Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg 370 375 380 Cys Thr Ala Thr Ser Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg 385 390 395 400 Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His 405 410 415 Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met 420 425 430 Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr Pro Ala Asp Ala Gly Thr 435 440 445 Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe 450 455 460 Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser 465 470 475 <210> 32 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> CS003 <400> 32 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile Gln 355 360 365 Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg 370 375 380 Cys Thr Ala Thr Ser Leu Val Pro Val Gly Pro Ile Gln Trp Phe Arg 385 390 395 400 Gly Ala Gly Pro Gly Arg Asn Leu Ile Tyr Asn Ser Arg His Gly His 405 410 415 Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met 420 425 430 Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr Pro Ala Asp Ala Gly Thr 435 440 445 Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe 450 455 460 Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser 465 470 475 <210> 33 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> CS004 <400> 33 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile Gln 355 360 365 Pro Asp Lys Ser Val Val Val Ala Ala Gly Glu Thr Ala Thr Leu Arg 370 375 380 Cys Thr Ala Thr Ser Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg 385 390 395 400 Gly Ala Gly Pro Gly Arg Ile Leu Ile Tyr Asn Arg Arg Gln Gly His 405 410 415 Phe Pro Arg Val Thr Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met 420 425 430 Asp Phe Ser Ile Arg Ile Gly Ala Ile Thr Pro Ala Asp Ala Gly Thr 435 440 445 Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ile Glu Phe 450 455 460 Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser 465 470 475 <210> 34 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> CS005 <400> 34 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile Gln 355 360 365 Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg 370 375 380 Cys Thr Ala Thr Ser Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg 385 390 395 400 Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His 405 410 415 Phe Pro Arg Val Thr Thr Val Ser His Ser Thr Lys Arg Asn Asn Met 420 425 430 Asp Phe Ser Ile Arg Ile His Ala Ile Thr Pro Ala Asp Ala Gly Thr 435 440 445 Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ser Glu Phe 450 455 460 Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser 465 470 475 <210> 35 <211> 479 <212> PRT <213> Artificial Sequence <220> <223> CS006 <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Pro 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Gly Ile Ser Thr Gly Gly Thr Thr Asn Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Leu Val Val Ser Gly Ile Gly Ser Thr Leu Glu Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile Gln 355 360 365 Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg 370 375 380 Cys Thr Ala Thr Ser Leu Phe Pro Val Gly Pro Ile Gln Trp Phe Arg 385 390 395 400 Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His 405 410 415 Phe Pro Arg Val Thr Thr Val Ser His Ser Thr Arg Arg Asn Asn Met 420 425 430 Asp Phe Ser Ile Arg Ile His Ala Ile Thr Pro Ala Asp Ala Gly Thr 435 440 445 Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ser Glu Phe 450 455 460 Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser 465 470 475

Claims

1. A fusion protein, which is composed of a first binding domain that specifically binds to CLDN18.2, a linker, and a second binding domain that specifically binds to CD47 protein. The first binding domain is an antibody targeting CLDN18.2 or an antigen-binding fragment thereof. The second binding domain is a mutant of human SIRPα variant 1, and the amino acid sequence of the mutant is shown in any one of SEQ ID NO: 2, 10 - 12, and 21.

2. The fusion protein according to claim 1, wherein the antibody is selected from the group consisting of: monoclonal antibody, chimeric antibody, humanized antibody, and fully human antibody.

3. The fusion protein according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab’, F(ab’)2, dAb, Fv, and scFv.

4. The fusion protein according to claim 1, wherein the CLDN18.2 comprises human CLDN18.

2.

5. The fusion protein according to claim 1, wherein the first binding domain is an immunoglobulin single variable domain.

6. The fusion protein according to claim 5, wherein the immunoglobulin single variable domain is VHH.

7. The fusion protein according to claim 6, wherein the amino acid sequence of the VHH is shown in SEQ ID NO:

24.

8. The fusion protein according to claim 1, wherein the first binding domain has a heavy chain constant region Fc fragment.

9. The fusion protein according to claim 8, wherein the heavy chain constant region is derived from IgG.

10. The fusion protein according to claim 9, wherein the heavy chain constant region is derived from human IgG.

11. The fusion protein according to claim 10, wherein the IgG is selected from the group consisting of: IgG1 and IgG4.

12. The fusion protein according to claim 8, wherein the amino acid sequence of the heavy chain constant region Fc fragment is shown in SEQ ID NO:

28.

13. The fusion protein according to claim 1, wherein the first binding domain is located at the N-terminus of the second binding domain.

14. The fusion protein according to claim 1, wherein the linker is located at the C-terminus of the first binding domain and at the N-terminus of the second binding domain.

15. The fusion protein according to claim 14, wherein the amino acid sequence of the linker is shown in SEQ ID NO:

29.

16. The fusion protein according to claim 1, which comprises at least 2 of the second binding domains.

17. The fusion protein according to claim 16, wherein each of the second binding domains is respectively located at the C-terminus of the first binding domain.

18. The fusion protein according to claim 1, which comprises at least one polypeptide chain, and the amino acid sequence of the polypeptide chain is shown in any one of SEQ ID NO: 30 - 35.

19. The fusion protein according to claim 1, which comprises two polypeptide chains, and the amino acid sequences of the two polypeptide chains are the same.

20. An immunoconjugate, which comprises the fusion protein according to any one of claims 1 - 19.

21. A nucleic acid molecule encoding the fusion protein according to any one of claims 1-19 or the immunoconjugate according to claim 20.

22. A vector comprising the nucleic acid molecule according to claim 21.

23. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-19, the immunoconjugate according to claim 20, the nucleic acid molecule according to claim 21, and optionally a pharmaceutically acceptable carrier.

24. A cell comprising the fusion protein according to any one of claims 1-19, the immunoconjugate according to claim 20, the nucleic acid molecule according to claim 21, or the vector according to claim 22.

25. A method for preparing the fusion protein according to any one of claims 1-19, which comprises culturing the cell according to claim 24 under conditions enabling the expression of the fusion protein.

26. Use of the fusion protein according to any one of claims 1-19, the immunoconjugate according to claim 20, the nucleic acid molecule according to claim 21, the vector according to claim 22, the pharmaceutical composition according to claim 23, or the cell according to claim 24 in the preparation of a medicament for preventing and / or treating a disease, which is gastric cancer, esophageal cancer, pancreatic cancer or lymphoma.

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