RU2800923C2 - Function protein and its use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to fusion proteins based on human SIRPα, and can be used in medicine for anticancer therapy or in the treatment of autoimmune diseases. A fusion protein for binding to the CD47 protein and a tumor-associated antigen is designed comprising the following: a first binding domain that specifically binds the tumor-associated antigen; and a second binding domain containing a human domain mutant SIRPα, which specifically binds the CD47 protein.

EFFECT: invention makes it possible to obtain a fusion protein capable of specifically binding both the CD47 protein and the tumor-associated antigen; and/or is capable of specifically blocking the interaction between the CD47 protein and SIRPα; and/or capable of effectively inhibiting the growth and/or proliferation of tumors or tumor cells.

20 cl, 16 dwg, 3 ex

Description

The technical field to which the invention belongs

The present application relates to the field of biomedicine, in particular to a multispecific fusion protein, as well as its use in the treatment of a tumor and/or an autoimmune disease.

State of the art invention

Currently, there are two main approaches in the field of tumor therapy: the introduction of targeted drugs and immunotherapy. These two therapies may interact with each other to produce a stronger cytotoxic effect, leading to sustained and long-term tumor reduction. However, the interaction between targeted drugs and immunotherapy is very complex, and the overall antitumor effect and toxicity profile of combination therapy can be influenced by various factors such as species, dosages, order, dosage forms, and the like.

The CD47 protein is a type of transmembrane glycoprotein that belongs to the immunoglobulin superfamily. In addition to being overexpressed by normal tissue cells, CD47 is overexpressed by many tumor cells. The CD47 protein on the surface of tumor cells binds to the SIRPα protein on the surface of macrophages, which prevents phagocytosis of tumor cells by macrophages, this is considered one of the mechanisms by which tumors avoid recognition by the immune system. Blocking the interaction between the CD47 protein and SIRPα can suppress tumor growth.

However, current reagents used to block the interaction between the CD47 protein and SIRPα have limited recognition activity, their affinity for the CD47 protein is always insufficient, so they have a limited ability to inhibit tumors. In addition, current CD47-targeting antibody drugs can cause side effects such as anemia reactions or thrombocytopenia. Therefore, it is imperative to obtain an effective therapy that specifically targets both the CD47 protein and its associated tumor antigens.

The essence of the invention

The present invention provides a fusion protein comprising a first binding domain that specifically binds a tumor associated antigen and a second binding domain that specifically binds a CD47 protein. The present invention also relates to an immunoconjugate comprising a fusion protein; a nucleic acid molecule encoding a fusion protein; a vector, composition, and cell capable of incorporating and/or expressing a fusion protein; and a method for producing a fusion protein. The fusion protein, immunoconjugate, nucleic acid molecule, vector, composition, and cell of the present invention have one or more of the following properties: 1) capable of specifically binding both the CD47 protein and a tumor-associated antigen; 2) are able to specifically block the interaction between the CD47 protein and SIRPα; 3) are capable of effectively inhibiting the growth and/or proliferation of tumors or tumor cells.

In one aspect, the present invention provides a fusion protein comprising: a first binding domain that specifically binds a tumor-associated antigen; and a second binding domain that specifically binds the CD47 protein; where the second binding domain contains a human SIRPα variant 1 mutant, this mutant contains a substitution, deletion or addition of an amino acid residue at one or more positions from site 33 to site 149 compared to the sequence shown in SEQ ID NO: 50.

In some embodiments, the mutant contains amino acid substitutions for one or more amino acid residues selected from the group consisting of: R22, I29, I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109 and V132.

In some embodiments, the mutant contains amino acid substitutions for amino acid residues selected from the group consisting of: (1) I61, V63, E77, E84, V93, L96, K98, N100, and V132; (2) I61, E77, Q82, K83 and E84; (3) I61, V63, K83, E84 and V132; (4) I61, E77, E84, R107 and V132; (5) I61, V63, E77, K83, E84 and N100; (6) I61, E77, Q82, K83, E84 and R107; (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109 and V132; (8) I61, E77, Q82, K83, E84 and V132; (9) I61; (10) I61, D95, L96, G109 and V132; (11) I61, D95, L96, K98, G109 and V132; (12) I61, E77, E84, V93, R107 and 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; and (20) I61, V63, E77, Q82 and E84.

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

In some embodiments, the mutant contains amino acid substitutions selected from the group consisting of: (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G, and V132L; (2) I61V, E77N, Q82S, K83R and E84H; (3) I61F, V63I, K83R, E84K and V132I; (4) I61L, E77Q, E84D, R107N and V132I; (5) I61L, V63I, E77K, K83R, E84D and N100G; (6) I61V, E77H, Q82R, K83R, E84H and R107S; (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R and V132R; (8) I61L, E77M, Q82G, K83R, E84D and V132L; (9) I61L; (10) I61F, D95H, L96S, G109H and V132S; (11) I61F, D95H, L96S, K98R, G109H and V132S; (12) I61L, E77Q, E84D, V93A, R107N and V132I; (13) E77K, L96S, N100K, G109H and V132L; (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D and V132I; (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D and V132L; (16) I61V, E77N, Q82S, K83R, E84H and V93A; (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R and N100E; (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D and G109R; (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R and N100G; and (20) I61L, V63I, E77N, Q82G and E84G.

In some embodiments, the mutant is the amino acid sequence shown in any of SEQ ID NOs: 51-70.

In some embodiments, the first binding domain comprises an antibody, or an antigen-binding fragment or variant thereof. In some embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody. In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, F(ab)2, dAb, isolated CDRs, Fv, and scFv.

In some embodiments, the antibody variant, or antigen-binding fragment thereof, is selected from the group consisting of:

a) a protein or polypeptide with a substitution, deletion or addition of one or more amino acids in the antibody or its antigen-binding fragment; And

b) a protein or polypeptide with at least 90% sequence homology with the antibody or antigen-binding fragment thereof.

In some embodiments, the tumor-associated antigen includes a tumor-associated antigen associated with a non-solid tumor and/or a solid tumor. In some embodiments, the tumor-associated antigen is selected from the group consisting of CD38, AXL, and Trop2.

In some embodiments, the first binding domain comprises an anti-CD38 antibody or antigen-binding fragment or variant thereof. In some embodiments, the CD38 is human CD38.

In some embodiments, the antibody comprises the heavy chain of the antibody or fragment thereof, the heavy chain of the antibody or fragment thereof comprises HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, sequentially. In some embodiments, the heavy chain of an antibody or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 8. In some embodiments, the antibody heavy chain or its fragment contains a heavy chain constant region, and the heavy chain constant region includes IgG. In some embodiments, the IgG is selected from the group consisting of IgG1 and IgG4. In some embodiments, the antibody heavy chain comprises any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21.

In some embodiments, the antibody comprises a light chain of an antibody or fragment thereof, the light chain of an antibody or fragment thereof comprises LCDR1-3, the amino acid sequences of LCDR1-3 are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, sequentially. In some embodiments, the light chain of an antibody or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 7. In some embodiments, the antibody light chain or its fragment contains a light chain constant region, and the light chain constant region includes Igκ. In some embodiments, the antibody light chain comprises any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.

In some embodiments, the first binding domain comprises an AXL antibody, or an antigen-binding fragment or variant thereof. In some embodiments, the AXL is a human AXL.

In some embodiments, the antibody comprises the heavy chain of the antibody or fragment thereof, the heavy chain of the antibody or fragment thereof comprises HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, sequentially. In some embodiments, the heavy chain of an antibody or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 29. In some embodiments, the antibody heavy chain or its fragment contains a heavy chain constant region, and the heavy chain constant region includes IgG. In some embodiments, the IgG is selected from the group consisting of IgG1 and IgG4. In some embodiments, the antibody heavy chain contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 34.

In some embodiments, the antibody comprises a light chain of an antibody or fragment thereof, the light chain of an antibody or fragment thereof comprises LCDR1-3, the amino acid sequences of LCDR1-3 are SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, sequentially. In some embodiments, the light chain of an antibody or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 28. In some embodiments, the antibody light chain or its fragment contains a light chain constant region, and the light chain constant region includes Igκ. In some embodiments, the antibody light chain contains an amino acid sequence selected from the group consisting of: SEQ ID NO: 32.

In some embodiments, the first binding domain comprises a Trop2 antibody, or an antigen-binding fragment or variant thereof. In some embodiments, Trop2 is human Trop2.

In some embodiments, the antibody comprises the heavy chain of the antibody or fragment thereof, the heavy chain of the antibody or fragment thereof comprises HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, sequentially. In some embodiments, the heavy chain of an antibody or fragment thereof comprises a VH heavy chain variable region, and the VH heavy chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 43. In some embodiments, the antibody heavy chain or its fragment contains a heavy chain constant region, and the heavy chain constant region includes IgG. In some embodiments, the IgG is selected from the group consisting of IgG1 and IgG4. In some embodiments, the heavy chain of the antibody comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 48. In some embodiments, the antibody contains a light chain of an antibody or fragment thereof, the light chain of an antibody or fragment thereof contains LCDR1-3, the amino acid sequences of LCDR1-3 are SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, sequentially. In some embodiments, the light chain of an antibody or fragment thereof comprises a VL light chain variable region, and the VL light chain variable region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 42. In some embodiments, an antibody light chain or its fragment contains a light chain constant region, and the light chain constant region includes Igκ. In some embodiments, the antibody light chain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 46.

In some embodiments, the first binding domain is located at the N-terminus of the second binding domain. In some embodiments, the fusion protein further comprises a linker, wherein the linker is located at the C-terminus of the first binding domain and is located at the N-terminus of the second binding domain. In some embodiments of the invention, the linker contains the amino acid sequence shown in any of SEQ ID NO: 73-74.

In some embodiments, the fusion protein contains at least two of the second binding domains. In some embodiments of the invention, each of the second binding domains is located at the C-terminus of the first binding domain, respectively.

In another aspect, the present invention relates to an immunoconjugate that contains a fusion protein.

In another aspect, the present invention provides one or more isolated nucleic acid molecules that encode a fusion protein or immunoconjugate.

In another aspect, the present invention relates to one or more vectors that contain nucleic acid molecules.

In another aspect, the present invention relates to a composition that contains a fusion protein, immunoconjugate or nucleic acid molecules and, optionally, pharmaceutically acceptable excipients.

In another aspect, the present invention relates to a cell that contains a fusion protein, immunoconjugate, nucleic acid molecules or vectors.

In another aspect, the present invention relates to a method for producing a fusion protein, which includes culturing a cell under conditions that allow expression of the fusion protein.

In another aspect, the present invention relates to the use of a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition or cell in the preparation of a drug, the drug being used to treat a tumor or an autoimmune disease.

In some embodiments, the tumor includes a non-solid tumor and a solid tumor.

In some embodiments, the tumor includes multiple myeloma, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, neuroglioma, germinoma, sarcoma, mesothelioma, placenta, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cavity cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, ovarian cancer, and testicular cancer.

In some embodiments of the invention, an autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin -dependent diabetes, miastenia, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, buffalo bubble syndrome, pemphigoid, and pemphigoid, pemphigoid, pemphigoid. h, multiple sclerosis, idiopathic polyneuritis , systemic lupus erythematosus, rheumatoid arthritis, scleroderma and polyarteritis nodosa.

In another aspect, the present invention relates to a fusion protein, immunoconjugate, nucleic acid molecule, vectors, composition or cell that are used to treat tumors or autoimmune diseases.

In another aspect, the present invention provides a method for blocking an interaction between a CD47 protein and SIRPα, comprising administering to a subject in need thereof an effective amount of a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition, or cell.

In another aspect, the present invention provides a method for inhibiting the growth and/or proliferation of tumors or tumor cells, comprising administering to a subject in need thereof an effective amount of a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition, or cell.

Other aspects and advantages of the present invention may be readily understood by those skilled in the art from the following detailed description. The following detailed description provides only examples of embodiments of the present invention. As will be appreciated by those skilled in the art, the content of this application allows those skilled in the art to make changes to the disclosed specific embodiments of the invention without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the accompanying drawings of the present application and the description of the present application are only exemplary and not restrictive.

Brief description of the drawings

Specific features of the present invention are given in the appended claims. The characteristics and advantages of the present invention will be better understood with reference to examples of embodiments of the invention and the accompanying drawings, detailed below. The accompanying drawings are briefly described below:

Figure 1 shows an exemplary structure of a fusion protein of the present invention.

Figures 2 and 3 show the biological activity of the fusion protein of the present invention.

Figures 4A and 4B show the biological activity of the fusion protein of the present invention.

Figure 5 is a diagram illustrating the principle of a method for detecting an interaction between a fusion protein of the present invention and CD38 and CD47.

Figure 6 shows the biological activity of the fusion protein of the present invention.

Figure 7 is a diagram illustrating the principle of a method for detecting an interaction between a fusion protein of the present invention and CD38 and CD47.

Figures 8 and 9 show the biological activity of the fusion protein of the present invention.

Figure 10 shows that the fusion protein of the present invention blocks the interaction between the CD47 protein and SIRPα.

Figures 11A-11B, Figures 12A-12B and Figures 13A-13B show the binding activity between the fusion protein of the present invention and the respective antigens.

Figure 14 shows tumor imaging results of mice in different treatment groups.

Figure 15 shows the average tumor fluorescence intensity of mice in different treatment groups.

Figure 16 shows the survival of mice in different treatment groups.

Detailed description of the invention

The implementation of the present invention will be illustrated in the following specific embodiments of the invention, and other advantages and effects of the present invention will be readily understood by those skilled in the art disclosed in the description.

In the present invention, the term "fusion protein" generally refers to a protein obtained by the fusion of two or more proteins or polypeptides. The fusion protein can be produced artificially using recombinant DNA technology. For example, genes or nucleic acid molecules encoding two or more proteins or polypeptides can be joined together to form fusion genes or fusion nucleic acid molecules that can encode a fusion protein. Translation of the fused genes may produce a single polypeptide, which may have properties of at least one or even each of the two or more pre-fusion proteins or polypeptides.

In the present invention, the term "specifically binds" generally refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antibody-producing antigen. One antibody specific for a certain antigen means binding to an antigen with an affinity (K _D )≤10 ^-5 M (for example, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M etc.), where the designation K _D refers to the ratio of the rate of dissociation to the rate of binding (k _off /k _on ), which can be determined by a method known to specialists in this field of technology.

In the present invention, the term "binding domain" generally means a domain that can specifically bind and/or recognize a particular epitope on a target (eg, antigen). In the present invention, the term "domain" generally refers to a structural region close to spherical in shape, which is clearly defined in the structure of protein subunits. For example, a polypeptide chain may first be a normal secondary structure formed from adjacent amino acid residues in some regions, then may also be a super secondary structure formed by assembling adjacent secondary structural fragments together, on this basis the polypeptide chain may be folded into a tertiary structure that has almost spherical shape. For larger protein molecules or subunits, the polypeptide chain can often be a tertiary structure that is formed by the association of two or more relatively independent regional structures that can be clearly distinguished in space, and such a relatively independent regional structure can be called a domain.

In the present invention, the terms "first", "second" used in the terms "first binding domain" and "second binding domain" are used only to distinguish in the description.

In the present invention, the term "CD47 protein" generally refers to an integrin-associated protein (IAP), which is a multiple transmembrane receptor belonging to the immunoglobulin superfamily. For example, the CD47 protein can bind to membrane integrins and also bind to their ligands thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα). The CD47 protein is highly expressed on the surface of the cell membrane. In the present invention, the CD47 protein may include any variants, isotypes, and species homologues of human CD47. The amino acid sequence of the human CD47 protein is listed as CEJ95640.1 in the GenBank database. The CD47 protein can be naturally expressed by cells or expressed on cells transfected with CD47 genes.

In the present invention, the term "SIRPα" generally refers to a regulatory membrane glycoprotein of the SIRP family that can be used as a ligand for the CD47 protein. In the present invention, SIRPα may include human SIRPα, for example, SIRPα variant 1 and SIRPα variant 2. SIRPα variant 2 differs from SIRPα variant 1 by 13 amino acids, and its amino acid sequence is listed as CAA71403.1 in the GenBank database. In the present invention, the term "SIRPα variant 1" generally refers to the SIRPα protein, the amino acid sequence of which is listed as NCBI RefSeq NP_542970.1 (residues 31-504 constitute the mature type), also the amino acid sequence of SIRPα Option 1 is shown in SEQ ID NO: 50 .

In the present invention, the term "variant" generally refers to a protein molecule that has sequence homology to the protein without any mutations/modifications, that retains at least a portion of the therapeutic and/or biological activity of the bioactive protein. For example, a protein variant may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity at compared to a reference bioactive protein. In some embodiments of the invention, the term "variant" may include proteins that have been intentionally modified (for example, through site-directed mutagenesis, synthesis, insertion, or sometimes mutation of coding genes).

The term "antibody" generally refers to a protein comprising one or more polypeptides predominantly encoded by immunoglobulin genes or immunoglobulin gene fragments. For example, immunoglobulin genes may include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. For example, a light chain can be classified into κ or λ, which can define immunoglobulin types, respectively: Igκ and Igλ. The heavy chain can be classified into γ, μ, α, δ or ε, which in turn determine the types of immunoglobulins, respectively: IgG, IgM, IgA, IgD and IgE. For example, an antibody may have structural units that include tetramers, each of the tetramers may consist of two pairs of identical polypeptide chains, and each pair has a "light chain" (about 25 kDa) and a "heavy chain" (about 50-70 kDa) , the N-terminus of each member may define a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. For example, the terms "light chain variable region (VL)" and "heavy chain variable region (VH)" generally refer to the light chain and heavy chain variable regions, respectively. The antibody may exist as a complete immunoglobulin or as a plurality of fully characterized fragments obtained by cleavage with various peptidases or by de novo expression.

In the present invention, the term "antigen-binding fragment" generally refers to one or more parts of a full-length antibody, in which the parts largely retain the ability to bind the same antigen (for example, CD38) to which the antibody binds, and they can compete with the full-length antibody for specific antigen binding. General theoretical information can be found in Fundamental Immunology, Ch. 7 (Paul, W., ed., Edition 2, Raven Press, N.Y. (1989), the entire contents of which are incorporated herein by reference. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of a complete antibody. B in some cases, the antigen-binding fragment contains Fab, Fab', F(ab')2, (Fab)2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), chimeric antibodies, diantibodies, and polypeptide which contains at least a portion of the antibody sufficient to render the polypeptide capable of specific binding to an antigen Conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or an enzymatic or chemical cleavage process) can be used to generate antigen-binding fragments antibodies from that antibody and screening antigen-binding fragments of the antibody for the presence of the same binding specificity as full-length antibodies. For example, pepsin can cleave antibodies in the hinge region below the disulfide bond to form F(ab')2.

In the present invention, the term "Fab" generally refers to antibody fragments consisting of VL, VH, CL and CH1 domains.

In the present invention, the term "Fab'" generally refers to antibody fragments with a few extra residues at the carboxyl end of the CH1 domain compared to Fab fragments. For example, Fab' may contain one or more cysteines derived from the hinge region of the antibody.

In the present invention, the term "F(ab)2" generally refers to antigen-binding fragments derived from cysteine-linked paired Fab fragments.

In the present invention, the term "dAb fragments" generally refers to antibody fragments consisting of VH domains (Ward et al., Nature 341: 544-546 (1989)).

In the present invention, the term "complementarity determining regions CDRs" generally refers to the 3 hypervariable regions (HVRs) of the light chain variable region (VL) and the heavy chain variable region (VH), and the hypervariable regions are also known as complementarity determining regions because these regions can form precise spatial complementarity with antigenic determinants.

In the present invention, the term "Fv fragments" generally refers to antibody fragments consisting of single chain VL and VH domains of an antibody.

In the present invention, the term "scFv" generally refers to molecules consisting of a heavy chain variable region and a light chain variable region of an antibody linked by a short peptide linker, also known as a single chain antibody.

In the present invention, the term "monoclonal antibody" generally refers to a group of antibodies that are substantially homogeneous, and the various antibodies contained in this group may be identical, except for the possible presence of trace amounts of naturally occurring mutations. Monoclonal antibodies are highly specific directly to one antigenic site. In addition, unlike a polyclonal antibody preparation that contains different antibodies for different determinants (epitopes), the "monoclonal" modifier of each monoclonal antibody for a single antigenic determinant is not interpreted as requiring the production of antibodies by any particular methods. For example, monoclonal antibodies can be generated by the hybridoma method, or monoclonal antibodies can be generated in bacterial, eukaryotic animal or plant cells using recombinant DNA technology, and can also be generated from a phage antibody library, for example, using the technology described in Clackson et al. ., Nature, 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).

As used herein, the term "chimeric antibody" generally refers to an antibody where a portion of each amino acid sequence of a heavy or light chain is homologous to a corresponding amino acid sequence in an antibody from a particular species or belongs to a particular category, and the remaining chain segments are homologous to corresponding sequences of another species. For example, the variable regions of the light chain and the heavy chain are derived from the variable regions of an antibody from one animal species (e.g., mice, rats, etc.), while the constant portion is homologous to the sequence of an antibody derived from another species (e.g., human) . For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to obtain variable regions, and the constant regions combined with them are derived from a person. Variable regions have the advantage of being easy to obtain and their specificity is not affected by the source of the constant regions associated with them. Meanwhile, since the constant regions of chimeric antibodies can be derived from a human, chimeric antibodies are less likely to elicit an immune response upon injection, compared with non-human constant region antibodies.

In the present invention, the term "humanized antibody" generally refers to a modified antibody that reduces the immunogenicity of an antibody, an immunoglobulin-binding protein and a polypeptide derived from a non-human species (e.g., mice or rats) in relation to a human, but retains the antigen-binding properties of the original antibody. For example, a humanized antibody can be generated using genetic engineering technology, and non-human binding domains can be humanized using the CDR transfer method (Jones et al., Nature 321:522 (1986)) and a variant thereof using technical means including reengineering ( Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), hyperchimerization (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154) and masking surface residues (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf B W, Dalton B J, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312). If other regions, such as the hinge region and constant region domains, also come from non-human sources, then these regions can also be humanized.

In the present invention, the term "fully human antibody" generally refers to an antibody obtained by expressing genes encoding a human antibody in a genetically engineered animal with a deletion of the antibody gene. For example, using transgenic or transchromosomal technology, all genes encoding a human antibody can be transferred to a genetically engineered animal with the antibody gene deleted so that the animal can express the human antibody.

Used in the present invention, the term "sequence homology" generally refers to the similarity of sequences or the possibility of interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When a computer program (e.g. Emboss Needle or BestFit) is used to determine sequence identity, similarity or homology between two different amino acid sequences, default settings can be used or an appropriate scoring matrix can be selected (e.g. blosum 45 or blosum 80) to optimize identity scores , similarities or homology. In some embodiments, homologous polynucleotides are those sequences that hybridize under stringent conditions, and they have a sequence identity of at least 60%, at least 65%, at least 70%, at least at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or even 100% between these sequences. When aligned with sequences of equivalent length, homologous polypeptides have a sequence identity of at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least measure. 98%, or may have sequence identity of at least 99%.

In the present invention, the terms "CD38", "CD38 protein" and "CD38 antigen" may be used interchangeably in this application and include any variants, isotypes and species homologues of human CD38 that can be naturally expressed by cells or is expressed on cells transfected CD38 genes. Synonyms for CD38 well known in the art include ADP-ribosylcyclase 1, cADPr hydrolase 1, Cd38-rs1, ADP cyclic ribose hydrolase 1, I-19, and NIM-R5 antigen. CD38 can be highly expressed on the surface of myeloma cells.

In the present invention, the terms "AXL", "AXL protein" and "AXL antigen" can be used interchangeably in this application and generally refer to a type of protein belonging to the tyrosine kinase family that has a common Gas6 ligand. In the present invention, high expression of AXL can be strongly associated with tumor formation, and AXL can be used as a marker of cancer progression. In the present invention, AXL may be human AXL, the amino acid sequence of which is in the GenBank database under accession number AAH32229.

In the present invention, the term "Trop2" generally refers to tumor-associated calcium signal transducer 2, which may be known as TACSTD2, EGP-1, GA733-1 or GP50, etc. Trop2 in cells can be localized to the cell membrane, and its positive staining sites can be mainly on the cell membrane. In normal tissues, Trop2 is minimally expressed, but overexpressed in tumor cells, so it can be used as a target for tumor therapy. In the present invention, Trop2 may be a human Trop2, the amino acid sequence of which is in the GenBank database under accession number NP_002344.

Typically, in a polypeptide chain, an amino group is linked to another carboxyl group in the polypeptide chain so as to become one chain, but amino acid residues remain at the two ends of the protein that do not form peptide bonds, respectively, which are the end of the polypeptide chain bearing free amino groups, and end of the polypeptide chain bearing carboxyl groups, respectively. In the present invention, the term "N-terminus" generally refers to the end of the polypeptide chain with an amino acid residue bearing free amino groups. In the present invention, the term "C-terminus" generally refers to the end of a polypeptide chain with an amino acid residue bearing free carboxyl groups.

In the present invention, the term "nucleic acid molecules" generally refers to nucleotides, deoxyribonucleotides or ribonucleotides or their analogues in isolated forms of any length, which are isolated from the natural environment or synthesized artificially.

As used herein, the term "immunoconjugate" generally refers to an immune function polypeptide molecule in which one or more heterogeneous molecules (including, without limitation, a cytotoxin) are conjugated. In the present invention, the terms "conjugate" and "bond", "fusion" can be used interchangeably in this application and generally refer to two or more chemical elements, sequences or components linked together, for example, by chemical conjugation or recombination . The heterogeneous molecule may be a cytotoxin, a chemotherapeutic agent, and so on. For example, a fusion protein of the present invention may be conjugated to one or more heterogeneous molecules (eg, a cytotoxin) to form an immunoconjugate.

In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a specific protein can be inserted so that the protein can be expressed. A vector causes an element of the genetic material it carries to be expressed in a host cell through transformation, transduction, or transfection of the host cell. For example, vectors include a plasmid; phagemid; cosmid; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-based artificial chromosome (PAC); phages such as λ phage or M13 phage; and animal viruses and the like. Varieties of animal viruses used as vectors include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus (eg, SV40). The vector may contain various elements to control expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain an origin of replication. The vector may also contain a component that helps it enter the cell, such as, but not limited to, a virion, a liposome, or a protein coat.

In the present invention, the term "tumor" generally refers to neoplasms resulting from the proliferation of local histocytes in mammalian organisms (eg, cells or parts thereof) under the influence of various oncogenic factors. In the present invention, a tumor may include a solid tumor and a non-solid tumor. Solid tumor may include neuroglioma, germinoma, sarcoma, mesothelioma, placenta, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, cancer prostate cancer, bowel cancer, breast cancer, cervical cancer, ovarian cancer and testicular cancer. In the present invention, a non-solid tumor may include multiple myeloma, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma.

In the present invention, the term "lymphoma" generally refers to a malignant tumor of the lymphatic system. Lymphoma may be due to lymph node cells or lymphocytes reproducing uncontrollably and producing cancer cells with abnormal abilities that can invade other tissues throughout the body. Signs and symptoms in patients may include lymphadenectasia, fever, night sweats, exhaustion or itching, and persistent fatigue. There are various subtypes of lymphoma, the two main being Hodgkin's lymphoma and non-Hodgkin's lymphoma. In the present invention, the term "Hodgkin's lymphoma (HL)" generally refers to lymphoma produced by lymphocytic leukocytes. About half of cases of Hodgkin's lymphoma are caused by Epstein-Barr viruses. In the present invention, the term "non-Hodgkin's lymphoma (NHL)" generally refers to other types of lymphoma, with the exception of Hodgkin's lymphoma.

In the present invention, the term "leukemia" generally refers to a malignant proliferative disease of the hematopoietic system, and generally refers to a type of disease caused by massive proliferation or accumulation of leukemic cells. Clonal leukemia cells proliferate and accumulate in the bone marrow and other hematopoietic tissues largely due to mechanisms such as uncontrolled proliferation, disdifferentiation and blocking of apoptosis, and infiltrate other non-hematopoietic tissues and organs, and at the same time suppress the normal hematopoietic function. Clinical manifestations are anemia of varying degrees, bleeding, infectious fever, as well as swelling of the liver, spleen, lymph nodes and skeletal pain.

In the present invention, the term "multiple myeloma" generally refers to a malignant tumor affecting the bone marrow caused by abnormal proliferation of plasma cells. Multiple myeloma causes the accumulation of cancer cells in the bone marrow, resulting in the removal of healthy hemocytes. Cancer cells produce abnormal proteins that can cause complications rather than normal antibodies. The incidence of multiple myeloma may increase in the elderly. In the treatment of multiple myeloma with chemotherapy, there is a low cell proliferation rate and strong drug resistance, which limits the chemotherapeutic efficacy. More than 90% of patients with multiple myeloma are resistant to chemotherapy.

In the present invention, the term "Raja cell" generally refers to continuous human cell lines that can produce strains of the Epstein-Barr virus. Viruses transform umbilical cord lymphocytes and induce early antigens in Raja's cells. Raja cells can be widely used as hosts for transfection and can also be used to detect hematopoietic cells and other malignant cellular neoplasms. In addition, Raja cells have and express several receptors for some complement components and immunoglobulin G Fc receptors, so they can also be used to detect immune complexes.

In the present invention, the term "autoimmune disease" generally refers to a disease caused by the body's immune response to self antigens resulting in damage to its own tissues. Autoimmune diseases can be divided into two main classes: organ-specific autoimmune diseases and systemic autoimmune diseases. Organ-specific autoimmune diseases may refer to pathological damage and dysfunction of tissues and organs, but their localization is limited to the specific organ targeted by antibodies or sensitized lymphocytes, and these diseases may include chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis. Systemic autoimmune diseases may be multiple organ lesions throughout the body due to extensive deposition of antigen-antibody complexes in the vascular wall or may be caused by cellulose-like necrotic inflammation of the vascular wall and interstitium and subsequent proliferation of collagen fibers in multiple organs caused by immune attack; and systemic autoimmune diseases may include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, and polyarteritis nodosa.

In the present invention, the term "ADCC" means "antibody-dependent cell-mediated cytotoxicity" (ADCC) and, as a rule, refers to the fact that cells with cytolytic activity, using Fc receptors (FcR) expressed on their surface, recognize the Fc segments located on the antigen -targets, due to which the effector cells of the immune system actively lyse target cells in which the membrane surface antigen has bound to a specific antibody.

In the present invention, the term "comprise" generally means the inclusion of well-defined features, but not the exclusion of other factors.

In the present invention, the term "about" generally refers to variations in the range of 0.5-10% above or below the specified value, for example, variations in the 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.

Fusion protein

In one aspect, the present invention provides a fusion protein, the fusion protein may comprise a first binding domain and a second binding domain. The first binding domain can specifically bind to a tumor-associated antigen; the second binding domain may specifically bind to the CD47 protein, the second binding domain may comprise a human SIRPα variant 1 mutant, and the mutant contains a substitution, deletion, or addition of amino acid residues in 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 from site 33 to site 149 compared to the sequence shown in SEQ ID NO: 50. The fusion protein of the present of the invention can specifically bind both tumor-associated antigen and CD47 protein, thereby playing a role in the treatment of tumors and/or autoimmune diseases.

In the present invention, the term "first binding domain" generally refers to a domain that can specifically bind a tumor-associated antigen. The term "second binding domain" generally refers to a domain that can specifically bind the CD47 protein.

In the present invention, a tumor-associated antigen may include a tumor-associated antigen associated with a non-solid tumor and/or a solid tumor. For example, the tumor associated antigen may be selected from the group consisting of CD38, AXL and Trop2.

Second binding domain specific to CD47

In the present invention, the mutant (e.g., the human SIRPα variant 1 mutant that specifically binds the CD47 protein) contains amino acid substitutions in 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 residue positions selected from the group consisting of: R22, I29, I61, V63, E77, Q82, K83, E84, V93, D95, D96, K98, N100, R107, G109 and V132.

In the present invention, the positions of amino acid residues in amino acid substitutions mean the number of residues determined based on the amino acid sequence shown in SEQ ID NO: 50.

In the present invention, "amino acid substitution Xn" means that the amino acid substitution occurs at the X residue of position n in the corresponding amino acid sequence as shown in SEQ ID NO: 50, where n is a positive integer, X is an abbreviation for any amino acid residue. For example, "amino acid substitution I61" indicates that the amino acid substitution occurs at residue I of position 61 in the corresponding amino acid sequence as shown in SEQ ID NO: 50.

In the present invention, amino acid substitutions may be non-conservative substitutions. Non-conservative substitutions may include changing amino acid residues in target proteins or polypeptides in a non-conservative manner, such as changing an amino acid residue with a particular side chain size or a particular property (e.g., hydrophilic) to another amino acid residue with a different side chain size or other property (e.g., , hydrophobic).

In the present invention, amino acid substitutions may also be conservative substitutions. Conservative substitutions may include changing amino acid residues in target proteins or polypeptides in a conservative manner, such as substituting an amino acid residue with a particular side chain size or property (e.g., hydrophilic) for another amino acid residue with the same or similar side chain size or the same or a similar property (eg, still hydrophilic). Such conservative substitutions usually do not greatly affect the structure or function of the resulting protein. In the present invention, a variant amino acid sequence of a fusion protein or fragment thereof may contain conservative amino acid substitutions that do not significantly alter the structure or function of the protein (e.g., a human SIRPα variant 1 mutant that blocks CD47 and specifically binds the CD47 protein).

By way of example, interchanges of various amino acids within each group of the following groups may be considered as conservative substitutions in the present invention: a group of amino acids with non-polar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine; a group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; a group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid; positively charged basic amino acids: lysine, arginine and histidine; and amino acids bearing phenyl groups: phenylalanine, tryptophan and tyrosine.

In the present invention, the mutant may contain amino acid substitutions for amino acid residues selected from the group consisting of: (1) I61, V63, E77, E84, V93, L96, K98, N100 and V132; (2) I61, E77, Q82, K83 and E84; (3) I61, V63, K83, E84 and V132; (4) I61, E77, E84, R107 and V132; (5) I61, V63, E77, K83, E84 and N100; (6) I61, E77, Q82, K83, E84 and R107; (7) I61, E77, Q82, E84, V93, L96, N100, R107, G109 and V132; (8) I61, E77, Q82, K83, E84 and V132; (9) I61; (10) I61, D95, L96, G109 and V132; (11) I61, D95, L96, K98, G109 and V132; (12) I61, E77, E84, V93, R107 and 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; and (20) I61, V63, E77, Q82 and E84.

In the present invention, the mutant may contain one or more (for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid substitutions selected from the group consisting of: R22C, I29L, I61L/V/F, V63I, E77I/N/Q/K/H/M/R/N/V/L, Q82S/R/G/N, K83R, E84K/H/D/R/G, V93L/A, D95H/R/E, D96S/T, K98R, N100G/K/D/E, R107N/S, G109R/H and V132L/R/I/ S.

In the present invention, the amino acid substitution "XnY/Z" means that the X residue at position n in the corresponding amino acid sequence as shown in SEQ ID NO: 50 is replaced by a Y amino acid residue or a Z amino acid residue, where n is a positive integer, X , Y, and Z are shorthand for any amino acid residues independently, and X is different from Y or Z. For example, the amino acid substitution "I61L/V/F" means that residue I at position 61 in the corresponding amino acid sequence as shown in SEQ ID NO: 50, replaced by amino acid residue L, V or F.

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

In the present invention, based on human SIRPα variant 1 (amino acid sequence shown in SEQ ID NO: 50, i.e. residues at positions 33 to 149 in the human SIRPα amino acid sequence), SIRPα variant 1 mutants containing amino acid substitutions of the above groups (1) - (20), respectively, are designated as M1, M5, M12, M35, M37, M41, M57, M67, M81, M82, M84, M91, M99, M102, M111, M122, M126, M130, M135 and M145 in series. SIRPα variant 1 mutants may sequentially contain the amino acid sequences shown in SEQ ID NO: 51 - SEQ ID NO: 70.

In some embodiments, the SIRPα variant 1 mutant is M91 and the SIRPα variant 1 mutant contains the amino acid sequence shown in SEQ ID NO: 62.

First binding domain that specifically binds a tumor-associated antigen

In the present invention, the first binding domain may comprise an antibody, or an antigen-binding fragment or variant thereof. For example, the antibody may be selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. For example, the antigen binding fragment is selected from the group consisting of: Fab, Fab', (Fab')2, F(ab)2, dAb, isolated complementarity determining regions CDR, Fv and scFv.

In the present invention, an antibody variant or antigen-binding fragment thereof may be a protein or polypeptide with one or more substitutions, deletions, or additions (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7 , 1-8, 1-9, 1-10 or more) amino acids in an antibody or antigen-binding fragment thereof. Alternatively, an antibody variant or antigen-binding fragment thereof may be a protein or polypeptide with at least 90% sequence homology (e.g., at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least at least 100%) with the antibody or its antigen-binding fragment.

First binding domain specific to CD38

An antibody or antigen-binding fragment or variant thereof according to the present invention can kill tumor cells and/or inhibit tumor growth by specifically binding to the CD38 protein. For example, the tumor may include a CD38 positive tumor. For example, a CD38 positive tumor may be selected from the group consisting of myeloma, lymphoma, and leukemia. In addition, for example, the tumor is selected from the group consisting of non-Hodgkin's lymphoma and Hodgkin's lymphoma. The tumor cells may be selected from the group consisting of Raji cells, Dowdy cells, Ramos cells and RPMI8226 cells. In the present invention, an antibody, antigen-binding fragment or variant thereof can kill multiple myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma cells or inhibit the growth of multiple myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma and Hodgkin's lymphoma.

The CD38 protein of the present invention may be a human CD38 protein or a functional fragment thereof. For example, a CD38 protein may not be a mouse CD38 protein or may not be a rat CD38 protein. In some embodiments, the antibody, antigen-binding fragment thereof, or variant of the present invention does not substantially bind to mouse CD38 protein or rat CD38 protein.

An antibody, antigen-binding fragment or variant thereof according to the present invention may compete with a reference antibody for binding to the CD38 protein. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of a reference antibody may comprise LCDR1-3, the LCDR1-3 amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, in sequence. The heavy chain variable region of the reference antibody may comprise HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, sequentially.

For example, the amino acid sequence of the light chain variable region of the reference antibody may be SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region of the reference antibody may be SEQ ID NO: 8. Further, for example, the light chain of the reference antibody may comprise any one of the amino acids sequences selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20; and the heavy chain of the reference antibody may comprise any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21. For example, the light chain of the reference antibody may contain the amino acid sequence shown in SEQ ID NO: 11, and the heavy chain of the reference antibody may contain the amino acid sequence shown in SEQ ID NO: 13. For example, the light chain of the reference antibody may contain the amino acid sequence shown in SEQ ID NO: 16, and a reference antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 17. For example, a reference antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 18 and a reference antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 19. For example, a reference antibody light chain may contain the amino acid sequence set forth in SEQ ID NO: 20 and a reference antibody heavy chain may contain the amino acid sequence set forth in SEQ ID NO: 21.

An antibody, an antigen-binding fragment thereof, or a variant of the present invention may comprise a light chain of an antibody or fragment thereof. For example, the light chain of an antibody or fragment thereof may contain an Igκ constant region, for example, may contain a human Igκ constant region.

For example, the light chain of an antibody or fragment thereof may contain LCDR1 and LCDR1 may contain the amino acid sequence as shown below in SEQ ID NO: 1. The light chain of an antibody or fragment thereof may contain LCDR2 and LCDR2 may contain the amino acid sequence as shown below in SEQ ID NO: 2. The light chain of an antibody or fragment thereof may contain LCDR3, and LCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 3 below.

The light chain of an antibody or fragment thereof of the present invention may comprise a VL light chain variable region, and the amino acid sequence of the VL light chain variable region may be SEQ ID NO: 7. In some embodiments, the light chain of an antibody or fragment thereof may comprise any one of the amino acids sequences selected from the group consisting of: SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20.

An antibody or antigen-binding fragment thereof of the present invention may comprise the heavy chain of an antibody or fragment thereof. For example, the heavy chain of an antibody or fragment thereof further comprises a human constant region. Wherein the human constant region may include a human IgG constant region. Wherein, the IgG constant region may include a human IgG1 or IgG4 constant region.

For example, the heavy chain of an antibody or fragment thereof may contain HCDR1 and HCDR1 may contain an amino acid sequence as shown in SEQ ID NO: 4 below. An antibody or fragment heavy chain may contain HCDR2 and HCDR2 may contain an amino acid sequence as shown below in SEQ ID NO: 5. In addition, for example, the heavy chain of an antibody or fragment thereof may contain HCDR3, and HCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 6 below.

The heavy chain of an antibody or fragment thereof may comprise a VH heavy chain variable region, and the VH heavy chain variable region may comprise an amino acid sequence as shown in SEQ ID NO: 8 below. In some embodiments, an antibody heavy chain may comprise any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 21.

In some embodiments of the invention in the antibody or antigennegative fragment of the present invention, the amino acid sequence of LCDR1 may contain SEQ ID NO: 1; the amino acid sequence of LCDR2 may comprise SEQ ID NO: 2; the amino acid sequence of LCDR3 may comprise SEQ ID NO: 3; and the amino acid sequence of HCDR1 may contain SEQ ID NO: 4; or the amino acid sequence of HCDR2 may contain SEQ ID NO: 5; the amino acid sequence of HCDR3 may comprise SEQ ID NO: 6. For example, an antibody or antigen-binding fragment thereof may comprise an SG003 antibody or an antibody having the same LCDR1-3 and HCDR1-3 as an SG003 antibody. In some embodiments, the antibody light chain or antigen-binding fragment thereof of the present invention may comprise a light chain variable region, the amino acid sequence of which may include SEQ ID NO: 7; and its heavy chain may comprise a heavy chain variable region, the amino acid sequence of which may comprise SEQ ID NO: 8. For example, an antibody or antigen-binding fragment thereof may comprise an SG003 antibody or an antibody having the same light chain variable region and heavy chain variable region such that and the SG003 antibody. In some embodiments, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain and a heavy chain, the amino acid sequence of the light chain is shown in SEQ ID NO: 11, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13. For example, an antibody or its the antigen binding fragment may comprise the SG003 antibody or have the same light chain and heavy chain amino acid sequences as the SG003 antibody.

In some embodiments, an antibody of the present invention may be SG003. The amino acid sequences of LCDR1-3 in the SG003 antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 7; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 8; the amino acid sequence of the light chain is shown in SEQ ID NO: 11; and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 13.

An antibody or antigen-binding fragment or variant thereof may also contain one or more random mutations (eg, one or more, eg one or more amino acid substitutions) in the light chain and/or heavy chain amino acid sequence of the SG003 antibody. For example, an antibody, antigen-binding fragment or variant thereof may contain one or more random mutations (e.g. one or more, e.g. one or more amino acid substitutions) at one or more L-FR1-4 framework region sites in the SG003 light chain variable region, and/or contain one or more random mutations (eg, one or more, eg, one or more amino acid substitutions) at one or more sites of the H-FR1-4 framework regions in the SG003 heavy chain variable region. For example, after random mutations, the light chain of an antibody or antigen-binding fragment or variant thereof may comprise any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20; and/or the heavy chain of an antibody, or an antigen-binding fragment thereof, or a variant may comprise any one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19. and SEQ ID NO: 21. Antibody to CD38 or an antigen-binding fragment or variant thereof, after random mutations, still has the ability to specifically bind the human CD38 protein.

In some embodiments, the amino acid sequence of the light chain of the antibody or antigen-binding fragment of the present invention contains SEQ ID NO: 11, and the amino acid sequence of the heavy chain contains SEQ ID NO: 13; alternatively, in an antibody or antigen-binding fragment thereof of the present invention, the light chain amino acid sequence comprises SEQ ID NO: 16 and the heavy chain amino acid sequence comprises SEQ ID NO: 17; alternatively, the amino acid sequence of the light chain of the antibody or antigen-binding fragment of the present invention contains SEQ ID NO: 18, and the amino acid sequence of the heavy chain contains SEQ ID NO: 19; alternatively, in an antibody or antigen-binding fragment thereof of the present invention, the light chain amino acid sequence is SEQ ID NO: 20 and the heavy chain amino acid sequence is SEQ ID NO: 21.

First binding domain specifically binding AXL

An antibody or antigen-binding fragment thereof or a variant of the present invention can kill tumor cells and/or inhibit tumor growth by specifically binding AXL. For example, AXL may be a human AXL.

An antibody, antigen-binding fragment thereof, or a variant of the present invention may compete with a reference antibody for binding to the AXL protein. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of a reference antibody may comprise LCDR1-3, the LCDR1-3 amino acid sequences are SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, sequentially. The heavy chain variable region of the reference antibody may comprise HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, sequentially.

For example, the amino acid sequence of the light chain variable region of the reference antibody may be SEQ ID NO: 28, and the amino acid sequence of the heavy chain variable region of the reference antibody may be SEQ ID NO: 29. Further, for example, the amino acid sequence of the light chain of the reference antibody may be is SEQ ID NO: 32; and the heavy chain amino acid sequence of the reference antibody may be SEQ ID NO: 34.

An antibody, antigen-binding fragment thereof, or a variant of the present invention may comprise a light chain of the antibody or fragment thereof. For example, the light chain of an antibody or fragment thereof may contain an Igκ constant region, for example, may contain a human Igκ constant region.

For example, the light chain of an antibody or fragment thereof may contain LCDR1 and LCDR1 may contain the amino acid sequence as shown in SEQ ID NO: 22 below. The light chain of an antibody or fragment thereof may contain LCDR2 and LCDR2 may contain the amino acid sequence as shown below in SEQ ID NO: 23. The light chain of an antibody or fragment thereof may contain LCDR3, and LCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 24 below.

The light chain of an antibody of the present invention, or a fragment thereof, may comprise a VL light chain variable region, and the amino acid sequence of the VL light chain variable region may be SEQ ID NO: 28. In some embodiments, the light chain amino acid sequence of an antibody or fragment thereof may be SEQ ID NO: 32.

An antibody or antigen-binding fragment thereof of the present invention may comprise the heavy chain of an antibody or fragment thereof. For example, the heavy chain of an antibody or fragment thereof further comprises a human constant region. Wherein the human constant region may comprise a human IgG constant region. Wherein the IgG constant region may comprise a human IgG1 or IgG4 constant region.

For example, the heavy chain of an antibody or fragment thereof may contain HCDR1 and HCDR1 may contain an amino acid sequence as shown in SEQ ID NO: 25 below. An antibody or fragment heavy chain may contain HCDR2 and HCDR2 may contain an amino acid sequence as shown below in SEQ ID NO: 26. In addition, for example, the heavy chain of an antibody or fragment thereof may contain HCDR3, and HCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 27 below.

The heavy chain of an antibody or fragment thereof may comprise a VH heavy chain variable region, and the amino acid sequence of the VH heavy chain variable region may be SEQ ID NO: 29. In some embodiments, the amino acid sequence of the heavy chain of an antibody or fragment thereof may be SEQ ID NO. : 34.

In some embodiments of the invention, the amino acid sequence of the LCDR1 antibody or antigennegative fragment of the present invention may contain SEQ ID NO: 22 or a variant thereof; the amino acid sequence of LCDR2 may comprise SEQ ID NO: 23 or a variant thereof; the amino acid sequence of LCDR3 may comprise SEQ ID NO: 24 or a variant thereof; and the amino acid sequence of HCDR1 may contain SEQ ID NO: 25 or a variant thereof; the amino acid sequence of HCDR2 may comprise SEQ ID NO: 26 or a variant thereof; the amino acid sequence of HCDR3 may comprise SEQ ID NO: 27 or a variant thereof. For example, an antibody or antigen-binding fragment thereof may comprise a C6G12 antibody or an antibody having the same LCDR1-3 and HCDR1-3 as a C6G12 antibody. In some embodiments, the light chain of an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region, and the amino acid sequence of the light chain variable region may comprise SEQ ID NO: 28 or a variant thereof; and its heavy chain may comprise a heavy chain variable region, and the amino acid sequence of the heavy chain variable region may comprise SEQ ID NO: 29 or a variant thereof. For example, an antibody or antigen-binding fragment thereof may comprise a C6G12 antibody or an antibody having the same light chain variable region and heavy chain variable region as a C6G12 antibody. In some embodiments, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain and a heavy chain, the amino acid sequence of the light chain is set forth in SEQ ID NO: 32, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 34. For example, an antibody or its the antigen binding fragment may comprise a C6G12 antibody or have the same light chain and heavy chain amino acid sequences as the C6G12 antibody.

In some embodiments, an antibody of the present invention may be a C6G12 antibody. the amino acid sequences of LCDR1-3 of the C6G12 antibody are shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 28; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 29; the amino acid sequence of the light chain is shown in SEQ ID NO: 32; and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 34.

First binding domain specific to Trop2

An antibody or antigen-binding fragment thereof or a variant of the present invention can kill tumor cells and/or inhibit tumor growth by specifically binding Trop2. For example, Trop2 may be human Trop2.

An antibody, antigen-binding fragment thereof, or a variant of the present invention may compete with a reference antibody for binding to the Trop2 protein. The reference antibody may comprise a light chain variable region and a heavy chain variable region. For example, the light chain variable region of a reference antibody may comprise LCDR1-3, the LCDR1-3 amino acid sequences are SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, sequentially. The heavy chain variable region of the reference antibody may comprise HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, sequentially.

For example, the amino acid sequence of the light chain variable region of the reference antibody may be SEQ ID NO: 42, and the amino acid sequence of the heavy chain variable region of the reference antibody may be SEQ ID NO: 43. Further, for example, the amino acid sequence of the light chain of the reference antibody may be is SEQ ID NO: 46; and the amino acid sequence of the heavy chain of the reference antibody may be SEQ ID NO: 48. For example, the light chain of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 46 and the heavy chain of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: :48.

An antibody, antigen-binding fragment thereof, or a variant of the present invention may comprise a light chain of the antibody or fragment thereof. For example, the light chain of an antibody or fragment thereof may contain an Igκ constant region, for example, may contain a human Igκ constant region.

For example, the light chain of an antibody or fragment thereof may contain LCDR1 and LCDR1 may contain the amino acid sequence as shown in SEQ ID NO: 36 below. The light chain of an antibody or fragment thereof may contain LCDR2 and LCDR2 may contain the amino acid sequence as shown below in SEQ ID NO: 37. The light chain of an antibody or fragment thereof may contain LCDR3, and LCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 38 below.

The light chain of an antibody or fragment thereof of the present invention may comprise a VL light chain variable region, and the VL light chain variable region may comprise an amino acid sequence as shown in SEQ ID NO: 42 below. In some embodiments, the light chain amino acid sequence of an antibody or fragment thereof may be SEQ ID NO: 46.

An antibody or antigen-binding fragment thereof of the present invention may comprise the heavy chain of an antibody or fragment thereof. For example, the heavy chain of an antibody or fragment thereof further comprises a human constant region. Wherein the human constant region may comprise a human IgG constant region. Wherein the IgG constant region may comprise a human IgG1 or IgG4 constant region.

For example, the heavy chain of an antibody or fragment thereof may contain HCDR1 and HCDR1 may contain an amino acid sequence as shown in SEQ ID NO: 39 below. An antibody or fragment heavy chain may contain HCDR2 and HCDR2 may contain an amino acid sequence as shown below in SEQ ID NO: 40. In addition, for example, the heavy chain of an antibody or fragment thereof may contain HCDR3, and HCDR3 may contain an amino acid sequence as shown in SEQ ID NO: 41 below.

The heavy chain of an antibody or fragment thereof may comprise a VH heavy chain variable region, and the VH heavy chain variable region may comprise an amino acid sequence as shown in SEQ ID NO: 43 below. In some embodiments, the amino acid sequence of an antibody heavy chain or fragment thereof may be is SEQ ID NO: 48.

In some embodiments of the invention in the antibody or antigennegative fragment of the present invention, the LCDR1 amino acid sequence may contain SEQ ID NO: 36 or a variant thereof; the amino acid sequence of LCDR2 may comprise SEQ ID NO: 37 or a variant thereof; the amino acid sequence of LCDR3 may comprise SEQ ID NO: 38 or a variant thereof; and the amino acid sequence of HCDR1 may contain SEQ ID NO: 39 or a variant thereof; the amino acid sequence of HCDR2 may comprise SEQ ID NO: 40 or a variant thereof; the amino acid sequence of HCDR3 may comprise SEQ ID NO: 41 or a variant thereof. For example, an antibody or antigen-binding fragment thereof may comprise an SG701 antibody or an antibody having the same LCDR1-3 and HCDR1-3 as an SG701 antibody. In some embodiments, the light chain of an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain variable region, the amino acid sequence of the light chain variable region may comprise SEQ ID NO: 42 or a variant thereof; and its heavy chain may contain a heavy chain variable region, the amino acid sequence of the heavy chain variable region may contain SEQ ID NO: 43 or a variant thereof. For example, an antibody or antigen-binding fragment thereof may comprise an SG701 antibody or an antibody having the same light chain variable region and heavy chain variable region as an SG701 antibody. In some embodiments, an antibody or antigen-binding fragment thereof of the present invention may comprise a light chain and a heavy chain, the amino acid sequence of the light chain is set forth in SEQ ID NO: 46, and the amino acid sequence of the heavy chain is set out in SEQ ID NO: 48. For example, an antibody or its the antigen binding fragment may comprise the SG701 antibody or have the same light chain and heavy chain amino acid sequences as the SG701 antibody.

In some embodiments, an antibody of the present invention may be an SG701 antibody. The amino acid sequences of the LCDR1-3 antibody SG701 are shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 42; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 43; the amino acid sequence of the light chain is shown in SEQ ID NO: 46; and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 48.

Association between the first binding domain and the second binding domain

In the present invention, 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 linked to the N-terminus of the second binding domain via a linker. In some cases, the C-terminus of the first binding domain may also be directly linked to the N-terminus of the second binding domain (eg, be in frame).

In the present invention, the fusion protein may also contain a linker, which may be located at the C-terminus of the first binding domain and located at the N-terminus of the second binding domain. For example, in a fusion protein, the C-terminus of a first binding domain may be linked to the N-terminus of a linker, and the C-terminus of a linker may be linked to the N-terminus of a second binding domain. For example, the first binding domain, the linker, and the second binding domain may be included sequentially from the N-terminus to the C-terminus of the fusion protein.

In the present invention, the linker may contain the amino acid sequence shown in any of SEQ ID NO: 73-74.

In some cases, the fusion protein may contain at least 2 (for example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more) second binding domains. In the present invention, each of the second binding domains may be located at the C-terminus of the first binding domain, respectively. In the present application, more than two second binding domains may be directly or indirectly linked to the C-terminus of the first binding domain.

In the present invention, the fusion protein may comprise a first binding domain that specifically binds the CD38 antigen and a second binding domain that specifically binds the CD47 protein, wherein the first binding domain may comprise an antibody that specifically binds the CD38 antigen or an antigen-binding fragment or variant thereof, the second binding the domain may contain a human SIRPα variant 1 mutant, the C-terminus of an antibody that specifically binds the CD38 antigen, or an antigen-binding fragment or variant thereof, may be directly or indirectly linked to the N-terminus of a human SIRPα variant 1 mutant. For example, the second binding domain may comprise at least two human SIRPα variant 1 mutants and the N-terminals of the two human SIRPα variant 1 mutants are linked to the C-termini of an antibody that specifically binds the CD38 antigen, or an antigen-binding fragment or variant thereof, respectively.

For example, as shown in Figure 1, the first binding domain of the fusion protein (SG3847L1) may contain SG003 and its second binding domain may contain two M91 variant 1 SIRPα mutants, the sequence of linker 1 used is given in SEQ ID NO: 73, N-termini of two M91 are linked to the C-terminus of two SG003 heavy chains via linker 1, respectively. In the fusion protein, M91 is linked to the C-terminus of the SG003 heavy chain to form a second polypeptide chain, and the SG003 light chain may be referred to as the first polypeptide chain. The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SG3847L1 are shown in SEQ ID NO: 75 and SEQ ID NO: 11, respectively.

For example, as shown in Figure 1, the first binding domain of the fusion protein (SG3847L2) may contain SG003, its second binding domain may contain two M91 variant 1 SIRPα mutants, the sequence of linker 2 used is shown in SEQ ID NO: 74, N-termini of two M91 linked to the C-terminus of two SG003 heavy chains via linker 2, respectively. In the fusion protein, M91 is linked to the C-terminus of the SG003 heavy chain to form a second polypeptide chain, and the SG003 light chain may be referred to as the first polypeptide chain. The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SG3847L2 are shown in SEQ ID NO: 76 and SEQ ID NO: 11, respectively.

In the present invention, the fusion protein may comprise a first binding domain that specifically binds the Trop2 antigen and a second binding domain that specifically binds the CD47 protein, wherein the first binding domain may comprise an antibody that specifically binds the Trop2 antigen, or an antigen-binding fragment or variant thereof, the second the binding domain may comprise a human SIRPα variant 1 mutant, and the C-terminus of an antibody that specifically binds the Trop2 antigen, or an antigen-binding fragment or variant thereof, may be directly or indirectly linked to the N-terminus of the human SIRPα variant 1 mutant. In the present invention, the second binding domain may comprise at least two human SIRPα variant 1 mutants and the N-termini of the two human SIRPα variant 1 mutants are linked to the C-termini of an antibody that specifically binds the AXL antigen, or an antigen-binding fragment or variant thereof, respectively. .

For example, as shown in Figure 1, the first binding domain of the fusion protein (SGTrop247) may contain the SG701 antibody, its second binding domain may contain two M91 variant 1 SIRPα mutants, the sequence of the linker used is shown in SEQ ID NO: 74, the N-termini of the two M91 linked to the C-terminus of two SG701 heavy chains via a linker, respectively. In the fusion protein, M91 is linked to the C-terminus of the heavy chain of SG701 to form a second polypeptide chain, and the light chain of SG701 may be referred to as the first polypeptide chain. The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGTrop247 are shown in SEQ ID NO: 78 and SEQ ID NO: 46, respectively.

In the present invention, the fusion protein may comprise a first binding domain that specifically binds the AXL antigen and a second binding domain that specifically binds the CD47 protein, where the first binding domain may comprise an antibody that specifically binds the AXL antigen, or an antigen-binding fragment or variant thereof, the second the binding domain may comprise a human SIRPα variant 1 mutant, the C-terminus of an antibody that specifically binds the AXL antigen, or an antigen-binding fragment or variant thereof, may be directly or indirectly linked to the N-terminus of a human SIRPα variant 1 mutant. In the present invention, the second binding domain may comprise at least two human SIRPα variant 1 mutants, and the N-terminals of the two human SIRPα variant 1 mutants are linked to the C-termini of an antibody that specifically binds the AXL antigen, or an antigen-binding fragment or variant thereof, respectively.

For example, as shown in Figure 1, the first binding domain of the fusion protein (SGAXL47) may contain the C6G12 antibody, its second binding domain may contain two M91 variant 1 SIRPα mutants, the sequence of the linker used is shown in SEQ ID NO: 74, N-termini of two M91 linked to the C-terminus of two C6G12 heavy chains via a linker, respectively. In the fusion protein, M91 is linked to the C-terminus of the C6G12 heavy chain to form a second polypeptide chain, and the C6G12 light chain may be referred to as the first polypeptide chain. The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGAXL47 are shown in SEQ ID NO: 77 and SEQ ID NO: 32, respectively.

Nucleic acid molecules, vectors and cells and methods for their production

In another aspect, the present invention provides one or more isolated nucleic acid molecules that encode a fusion protein or immunoconjugate. For example, each nucleic acid molecule of one or more nucleic acid molecules may encode an entire antibody or an antigen-binding fragment thereof, and may also encode a portion of it (for example, one or more of HCDR1-3, LCDR1-3, VL, VH, light chains, or heavy chains).

The nucleic acid molecules of the present invention can be isolated. For example, they can be prepared or synthesized by the following methods: (i) by in vitro amplification, e.g., preparation by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) by purification, e.g., fractionated by enzymatic digestion and gel electrophoresis, or (iv) by synthesis, for example by chemical synthesis. In some embodiments, the isolated nucleic acids are nucleic acid molecules obtained using recombinant DNA technology.

Recombinant DNA technology and molecular cloning techniques include those described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and in 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 also in Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Briefly, nucleic acids can be obtained from fragments of genomic DNA, cDNA and RNA, and all of these nucleic acids can be extracted from cells directly or obtained by recombination using various amplification methods (including, without limitation, PCR and RT-PCR).

Direct chemical synthesis of nucleic acids usually involves the sequential addition of 3'-capped and 5'-capped nucleotide monomers to the terminal 5'-hydroxyl of the growing nucleotide polymer chain, in which each addition is carried out by nucleophilic attack of the terminal 5'-hydroxyl of the growing chain in 3 '-position of added monomers, and the monomers are usually phosphorus derivatives such as phosphotriester, phosphoramidite, etc. See, for example, Matteuci et al, Tet. Lett. 521: 719 (1980); US Patent No. 4500707 Caruthers et al; and US Patents Nos. 5436327 and 5700637 by Southern et al. In another aspect, the present invention provides vectors comprising the isolated polynucleotide of the present invention. Vectors can be any linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and so on. Non-limiting examples of viral vectors may include retroviruses, adenoviruses, and adeno-associated viruses. In some embodiments, the vectors are expression vectors, such as phage display vectors.

In another aspect, the present invention relates to one or more vectors, including nucleic acid molecules. For example, the vector may contain one or more nucleic acid molecules of the present invention. Each vector may contain one or more nucleic acid molecules. In addition, the vector may additionally contain other genes, such as marker genes, which allow selection of the vector in suitable host cells and under appropriate conditions. In addition, the vector may further contain expression control elements to ensure correct expression of the coding regions in the appropriate host. Such controls are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other controls for regulating gene transcription or mRNA translation, and the like. In some embodiments, the expression control sequences are adjustable elements. The specific structures of expression control sequences may vary depending on the species or function of cell types, but typically include 5' non-transcribed sequences and 5' and 3' non-translated sequences that are involved in transcription initiation and translation, respectively, e.g. TATA cassettes, capped sequences , CAAT sequences, etc. For example, a 5' non-transcribed expression control sequence may contain a promoter region, which may contain a promoter sequence for transcription and control of operably linked nucleic acids. The expression control sequence may further comprise an enhancer sequence or a precursor activator sequence. In the present invention, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, the human U6RNA promoter, the CMV promoter, and artificial hybrid promoters (e.g., CMV) in which a certain portion of the promoter can be fused to a certain portion of the gene promoter. other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) and may or may not contain additional introns. One or more nucleic acid molecules of the present invention may be operably linked to expression control elements.

Vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used, for example, in genetic engineering. In some embodiments, the vectors may be expression vectors.

The vectors may also contain one or more selectable marker genes which, once expressed, can confer one or more phenotypic traits that can be used to select or identify host cells carrying the vectors in other ways. Non-limiting examples of suitable eukaryocyte selectable markers include resistance to dihydrofolate reductase and neomycin.

In another aspect, the present invention relates to a cell that contains a fusion protein, immunoconjugate, nucleic acid molecules or vectors. The cell may be a host cell. For example, a cell may include various cell types as follows: prokaryotic cells such as Escherichia coli or Bacillus subtilis , fungal cells such as yeast cells or Aspergillus , insect cells such as Drosophila or Sf9 S2 cells, or animal cells. such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

For example, vectors can be introduced into host cells stably or transiently using a variety of well known technologies. For example, one method includes calcium chloride treatment, in which the vectors are introduced by calcium precipitation. Other salts, such as calcium phosphate, may also be used in similar processes. In addition, electroporation (ie, the application of an electric current to increase the permeability of cells to nucleic acids) can be used. Other examples of transformation methods include microinjection, DEAE-mediated dextran transformation, and heat shock in the presence of lithium acetate. Lipid complexes, liposomes and dendrimers can also be used to transfect host cells.

When heterogeneous sequences are introduced into host cells, various methods can be used to identify host cells into which vectors have been introduced. One example of a selection method includes subculturing one cell to form a single colony and then testing for expression of the desired protein product. Another method involves the selection of host cells containing heterogeneous sequences, based on phenotypic traits due to the expression of selective marker genes included in the vectors.

For example, the introduction of various heterogeneous sequences of the present invention into host cells can be confirmed by the following methods, such as PCR, Southern blot or Northern blot. For example, nucleic acids can be isolated from the resulting host cells, and specific target sequences can be amplified by PCR using primers specific for the target sequences. The amplified products are subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, then stained with ethidium bromide, SYBR Green solution or the like, or DNA is detected by UV detection. Alternatively, nucleic acid probes specific for target sequences can be used in hybridization reactions. The expression of particular gene sequences can be determined by hybridization by PCR or Northern blotting, or by detection of reverse transcription of the corresponding mRNA using an immunoassay of antibodies that react with encoded gene products. Examples of immunoassays include, without limitation, solid phase ELISA, radioimmunoassay, and sandwich immunoassay.

In addition, the introduction of various heterogeneous sequences of the present invention into host cells can be confirmed by the enzymatic activity of enzymes (eg, enzyme markers) that are encoded by heterogeneous sequences. Enzymes can be determined by various methods known in the art. Usually, enzymatic activity can be determined by the formation of products or by the transformation of the substrate of the enzymatic reaction under study. The reaction can be carried out in vitro or in vivo .

In another aspect, the present invention relates to a method for producing a fusion protein, which may include culturing the cell under conditions that allow expression of the fusion protein. For example, suitable culture media, suitable culture temperature and time can be used, all methods known to those skilled in the art.

In some cases, the method may further include the steps of separating and/or purifying the fusion protein. For example, a fusion protein of the present invention can be purified and separated by affinity chromatography using protein G-agarose or protein A-agarose, or by gel electrophoresis and/or high performance liquid chromatography.

Immunoconjugate composition and use

In another aspect, the present invention provides an immunoconjugate comprising a fusion protein. For example, the immunoconjugate may be a fusion protein-drug conjugate (ADC) wherein the fusion protein of the present invention 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 immune inhibitors (eg, any agents that kill cells by inhibiting immune responses); and the like. In some embodiments, the therapeutic agents may be an agent capable of treating tumor-associated diseases or disorders.

Conjugation may be by peptide linkers (eg, cleavable linkers) or by other means. For example, the linkers can be acid labile linkers, peptidase sensitive linkers, photolabile linker, and the like.

In another aspect, the present invention relates to a composition comprising a fusion protein, immunoconjugate or nucleic acid molecules, and optionally pharmaceutically acceptable excipients.

For example, pharmaceutically acceptable excipients may include buffering agents, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugar, chelating agents, counterions, metal complexes, and/or nonionic surfactants, and the like. .

In the present invention, the composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known excipients and excipients using conventional techniques known in the art, for example, according to the technology described in Remington: The Science and Practice of Pharmacy, Edition 19, Gennaro ed., Mack Publishing Co., Easton, PA, 1995.

In the present invention, the composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at tumor sites, administration by inhalation, rectal administration, vaginal administration, transdermal administration, or administration using a subcutaneous reservoir.

For example, the composition may be used to inhibit tumor growth. For example, a composition of the present invention can inhibit or delay the development or progression of diseases, shrink tumors (even substantially eliminate tumors), and/or alleviate and/or stabilize disease states.

For example, the composition of the present invention may be in suitable forms 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 application in the form of an ointment or cream; or for rectal administration in the form of suppositories. The composition may be in unit dosage forms suitable for single administration in precise doses. The composition may further contain conventional drug carriers or excipients. In addition, the composition may contain other drugs or agents, carriers, adjuvants, etc.

The composition of the present invention may contain a therapeutically effective amount of a fusion protein. A therapeutically effective amount is the dose required to prevent and/or treat (at least partially cure) diseases or disorders (e.g., tumors) and/or any of their complications in subjects suffering from or at risk of these diseases or disorders. . The specific amount/concentration of the dose may vary depending on the route of administration and the needs of the patients and may be determined, for example, based on the size of the patients, viscosity and/or body weight. For example, a suitable dose may be from about 0.1 mg or from 1 mg/kg/day to about 50 mg/kg/day; sometimes the dose may be higher. It should be understood that these specific dosages can be readily adjusted by those skilled in the art (eg, physicians or pharmacists) depending on particular patients, drugs, and/or disease status.

In the present invention, the terms "treatment" or "cure" or "alleviation" or "improvement" can be used interchangeably in this application and refer to those methods that allow you to get useful or desired results (including, without limitation, therapeutic and / or prophylactic benefit). In the present invention, the term "therapeutic benefit" generally refers to the elimination or reduction of the severity of the underlying conditions being treated. In addition, therapeutic benefits are realized by eliminating or reducing the severity of underlying conditions or reducing the frequency of one or more physical symptoms associated with underlying conditions in order to observe improvements in subjects (although subjects may still suffer from underlying conditions). With regard to prophylactic benefit, the composition may be administered to subjects at risk of developing a particular disease, or to subjects with one or more physical symptoms of a known disease, even if the disease may not have been diagnosed.

In another aspect, the present invention relates to the use of a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition or cell for drug production, where the drug can be used to treat a tumor or an autoimmune disease.

In another aspect, a fusion protein, immunoconjugate, nucleic acid molecule, vectors, composition or cell of the present invention may be used to treat a tumor or an autoimmune disease.

In another aspect, the present invention relates to a method of treating a tumor or an autoimmune disease comprising administering to a subject a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition or cell of the present invention.

In another aspect, the present invention provides a method for blocking an interaction between a CD47 protein and SIRPα, comprising administering (eg, administering to a subject or cells or biological samples in need thereof) a fusion protein or composition of the present invention.

In another aspect, the present invention relates to a method for inhibiting a tumor or the growth and/or proliferation of tumor cells, comprising bringing a fusion protein or composition of the present invention into contact with a tumor or tumor cells. For example, the contact may be in vitro .

In some embodiments, the tumor may include a non-solid tumor and a solid tumor.

In some embodiments, the tumor may include multiple myeloma, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, neuroglioma, germinoma, sarcoma, mesothelioma, placenta, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer , esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, ovarian cancer, and testicular cancer.

In some embodiments, the autoimmune disease may include chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma and polyarteritis nodosa.

In another aspect, the present invention relates to a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition or cell that can be used to treat a tumor or an autoimmune disease.

In another aspect, the present invention provides a method for blocking an interaction between a CD47 protein and SIRPα, comprising administering to a subject in need thereof an effective amount of a fusion protein, immunoconjugate, nucleic acid molecules, vectors, composition, or cell.

In the present invention, the term "subject" generally refers to a human or non-human animal, including, without limitation, a cat, dog, horse, pig, cow, sheep, goat, rabbit, mouse, rat, or monkey.

In another aspect, the present invention relates to a method capable of inhibiting tumors or the growth and/or proliferation of tumor cells, which may include administering to a subject in need thereof an effective amount of a fusion protein, immunoconjugate, nucleic acid molecules, vectors, compositions or cells.

Without wishing to be bound by any theory, the following embodiments of the invention are only intended to illustrate the fusion protein, production method and use of the present invention, and not to limit the scope of the present invention.

Embodiments of the invention

Embodiment 1. Effect of different linker selection on activity

1.1 Preparation of a fusion protein using various linkers

According to the structure of the fusion protein shown in Figure 1, taking for example the humanized anti-CD38 antibody SG003 and the M91 mutant (SEQ ID: NO 62) of SIRPα variant 1, different linkers were used from the N-terminus to the C-terminus to sequentially bind SG003 , a linker, and two M91s, where the N-terminus of the two M91s are linked to the C-terminus of the SG003 heavy chain, respectively, to investigate the effect of different linkers on the biological activity of the fusion protein.

The amino acid sequences of the LCDR1-3 antibody SG003 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 7; the nucleotide sequence encoding VL is shown in SEQ ID NO: 9; the amino acid sequences of the HCDR1-3 antibody SG003 are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 8; the nucleotide sequence encoding VH is shown in SEQ ID NO: 10. The light chain amino acid sequence of antibody SG003 is shown in SEQ ID NO: 11; and the nucleotide sequence encoding it is shown in SEQ ID NO: 12. The amino acid sequence of the heavy chain of antibody SG003 is shown in SEQ ID NO: 13; and the nucleotide sequence encoding it is shown in SEQ ID NO: 14.

Linker 1 (SEQ ID: NO 73) was chosen and the engineered fusion protein was named SG3847L1. SG3847L1 consists of a first polypeptide chain and a second polypeptide chain. The first polypeptide chain is a light chain SG003, the amino acid sequence of which is shown in SEQ ID NO: 11; the second polypeptide chain is a polypeptide chain formed by linkage between the SG003 heavy chain and M91 through linker 1, the amino acid sequence of which is shown in SEQ ID NO: 75.

Linker 2 (SEQ ID: NO 74) was chosen and the engineered fusion protein was named SG3847L2. SG3847L2 consists of a first polypeptide chain and a second polypeptide chain. The first polypeptide chain is a light chain SG003, the amino acid sequence of which is shown in SEQ ID NO: 11; the second polypeptide chain is a polypeptide chain formed by linkage between the SG003 heavy chain and M91 through linker 2, the amino acid sequence of which is shown in SEQ ID NO: 76.

1.2 Analysis of the binding activity of two antigens, respectively

(1) Using SG003 as a control, the binding activity between the fusion protein and CD38 was assessed by ELISA.

Target antigen CD38-His (1 μg/ml) was applied to ELISA plates, incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; various concentrations of SG003 antibody, SG3847L1 or SG3847L2 fusion protein were added and reacted at 37°C for 1 hour; after washing with PBS, horseradish peroxidase-labeled secondary goat anti-human IgG (Goat Anti human IgG HRP, Thermo Fisher Scientific) was added, reacted at 37°C for 30 minutes, and washed with PBS 5 times; 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-2 minutes; then 100 μl of 2N H ₂ SO ₄ stop solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the ability of the fusion protein to bind the target CD38 antigen was analyzed.

As shown in Figure 2, compared with the SG003 antibody, the ability of SG3847L1, SG3847L2 to bind to the target CD38 antigen was similar, the EC ₅₀ values for SG3847L1, SG3847L2 and SG003 were 0.175 nM, 0.149 nM and 0.133 nM, respectively.

(2) Using SS002M91 as a control, the binding activity between the fusion protein and CD47 was assessed by ELISA.

M91 was fused with IgG1-FC (sequence shown in SEQ ID NO: 71) to give SS002M91, the amino acid sequence of which is shown in SEQ ID NO: 72.

Target antigen CD47-His (1 μg/ml) was applied to ELISA plates, incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; various concentrations of SS002M91, SG3847L1, or SG3847L2 were added and reacted at 37°C for 1 hour; after washing with PBS, horseradish peroxidase-labeled secondary goat anti-human IgG (Goat Anti human IgG HRP, Thermo Fisher Scientific) was added, reacted at 37°C for 30 minutes, and washed with PBS 5 times; 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-2 minutes; then 100 μl of 2N H ₂ SO ₄ stopping solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the ability of the fusion protein to bind the target CD47 antigen was analyzed.

As shown in Figure 3, compared to SS002M91, the ability of SG3847L1, SG3847L2 to bind to the target CD47 antigen was similar, the EC ₅₀ values for SG3847L1, SG3847L2 and SS002M91 were 0.114 nM, 0.091 nM and 0.072 nM, respectively.

(3) Using SG003 and SS002M91, respectively, as a control, the binding activity between the fusion protein and CD38 and CD47 antigens was assessed by ELISA, respectively.

CHO-S cell lines that were engineered to express human CD38 or human CD47 were used to evaluate the ability of the fusion protein to bind antigens on the cell surface. Cells in the period of logarithmic growth were collected, brought to a cell density of 5 × 10 ⁶ cells/ml and pre-cooled on ice. SG003, SS002M91, negative control antibody (IgG1), SG3847L1 or SG3847L2 were diluted to various concentrations with pre-chilled saline containing 2% FBS.

100 μl of cells were taken and added to an equal volume of the above diluted antibody or fusion protein and reacted in the dark at 4°C for 30 minutes. At the end of the reaction, the cells were washed twice. Cells were resuspended in 100 µl of diluted phycoerythrin labeled secondary goat anti-human IgG Fc (eBioscience) and reacted in the dark at 4°C for 30 min. At the end of the reaction, the cells were washed twice. Cells were resuspended in 400 μl of 1% paraformaldehyde. The binding capacity between the fusion protein and CD38 or CD47 on the cell surface was analyzed on a flow cytometer (BD Calibur).

As shown in Figures 4A and 4B, the designations "1", "2", "3", "4", "5", "6", "7", "8" and "9" refer to 20 μg ml ^{- 1} IgG1, 20 µg ml ^-1 SG003, 20 µg ml ^-1 SS002M91, 20 µg ml ^-1 SG3847L1, 10 µg ml ^-1 SG3847L1, 5 µg ml ^-1 SG3847L1, 20 µg ml ^-1 SG3847L2, 10 µg ml ^-1 SG3847L2 and 5 µg ml ^-1 SG3847L2, respectively. The results showed that the fusion proteins SG3847L1 and SG3847L2 can specifically recognize the CD38 or CD47 antigen expressed on the cell surface.

1.3 Analysis of the activity of simultaneous binding of two antigens

(1) Following the principle shown in Figure 5, using the humanized anti-CD38 antibody SG003 as a control, the biological activity of the SG3847L1 and SG3847L2 fusion proteins to simultaneously bind the two CD38 and CD47 antigens was analyzed by solid phase ELISA.

Target antigen CD38-His (1 μg/ml) was applied to ELISA plates, incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; various concentrations of SG003 antibody, SG3847L1 or SG3847L2 fusion protein were added and reacted at 37°C for 1 hour; and reacted at 37°C for 1 hour; after washing with PBS, biotin-labeled CD47 (Biotin-Fc-CD47) was added, the reaction was carried out at a temperature of 37°C for 30 minutes, and PBS was washed 5 times; horseradish peroxidase-labeled avidin (Streptavidin-HRP, Jiaxuan Bio.) was added, reacted at 37°C for 30 minutes, and washed with PBS 5 times; 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-2 minutes; then, 100 μl of 2N H ₂ SO ₄ stopping solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the ability of the fusion proteins to simultaneously bind the target CD38 and CD47 antigens was analyzed.

As shown in Figure 6, compared to the SG003 antibody, the fusion proteins SG3847L1, SG3847L2 could simultaneously bind the target antigens CD38 and CD47, and the EC50 values for SG3847L1 and SG3847L2 were 0.020 nM and 0.024 nM, respectively.

(2) According to the principle shown in Figure 7, using SG003 and SS002M91 as controls, the biological activity of the fusion proteins SG3847L1 and SG3847L2 was evaluated by simultaneously binding the two antigens CD38 and CD47 on the cell surface.

CHO-S cell lines that were engineered to express human CD38 were used to evaluate the ability of the fusion proteins to simultaneously bind two cell surface antigens. Cells in the period of logarithmic growth were collected, brought to a cell density of 5 × 10 ⁶ cells/ml and pre-cooled on ice. SG003 antibody, SS002M91 fusion protein, negative control antibody (IgG1), SG3847L1 or SG3847L2 was diluted with pre-chilled saline containing 2% PBS. 100 µl of cells were taken and added to an equal volume of the above diluted antibodies or fusion proteins and CD47-FITC (Jiaxuan Bio.) and reacted in the dark at 4°C for 30 minutes. At the end of the reaction, the cells were washed twice. Cells were resuspended in 100 µl of diluted phycoerythrin (PE) labeled secondary goat anti-human IgG Fc (eBioscience), i.e. Fc-PE, and reacted in the dark at 4°C for 30 min. At the end of the reaction, the cells were washed twice. Cells were resuspended in 400 μl of 1% paraformaldehyde. The ability of the fusion proteins to simultaneously bind CD38 and CD47 on the cell surface was analyzed on a flow cytometer (BD Calibur).

As shown in Figure 8, the fusion proteins SG3847L1 and SG3847L2 could simultaneously recognize CD38 and CD47 antigens on the cell surface, and SG3847L2 had a higher ability to simultaneously recognize two antigens.

1.4 Analysis of ADCC activity to kill CD38-positive cells

First, the target cells (Raja cells) required for the experiment were brought to a density of 2 × 10 ⁵ cells/ml, resuspended in ADCC buffer (MEM culture solution without phenol red + 1% FBS) and added to a 96-well plate (50 µl per well). Then, 100 μl of various concentrations of SG003, SG3847L1 and SG3847L2 was added to each well, mixed until homogeneous and then incubated in an incubator at 37°C and 5% CO ₂ for 30 minutes. Then, NK92MI-CD16a effector cells (Huabo Bio.) required for the experiment were adjusted to a density of 1.2×10 ⁶ cells/mL and added to the wells containing target cells so that the ratio of target cells to effector cells was 1 :6. After mixing until homogeneous, they were incubated in an incubator at 37°C and 5% CO ₂ for 6 hours. Then, 100 μl per well of the stock solution was removed from the 96-well plate, and 100 μl per well of the LDH reaction mixture from the Cytotoxicity Detection Kit (Roche) was added and the reaction was carried out at a temperature of 37°C for 10 minutes. An additional 50 µl per well of stop solution was added and mixed gently. OD at 492 nm was measured on a microplate reader and OD at 650 nm was also measured as background. The following control groups were formed simultaneously at the beginning of the experiment, where control 1 was ADCC buffer, control 2 was target cells + ADCC buffer, control 3 was target cells + lysate + ADCC buffer, control 4 was cells -targets + effector cells + ADCC buffer solution. Specific kill rate % = ((experimental group - control 4) / (control 3 - control 2)) × 100%. Data analysis was performed on a dose-response curve using GraphPad Prism version 5 software.

As shown in Figure 9, when Raja target cells and NK92MI-CD16a effector cells were used for detection, the SG3847L1 and SG3847L2 fusion proteins showed the best ADCC effect. EC ₅₀ values for SG003, SG3847L1, and SG3847L2 were 0.067 nM, 0.014 nM, and 0.013 nM, respectively.

1.5 Activity assay for blocking the interaction between CD47 protein and SIRP α

Using SS002M91 as a control, the biological activity of the fusion proteins SG3847L1 and SG3847L2 was evaluated to block the interaction between the CD47 protein and SIRPα.

SIRPα-His was applied to the assay plate at a concentration of 1 μg/ml and incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; SS002M91, SG3847L1 or SG3847L2 were gradiently diluted with 10% fetal calf blood, respectively, and biotin-Fc-CD47 was added as primary antibody to the samples to a final concentration of 2 μg/ml, pre-incubated at 37°C for 30 minutes; after washing the analytical plate with PBST, the primary antibody was added and incubated at 37°C for 1 hour; after washing 5 times with PBS, horseradish peroxidase labeled avidin (Streptavidin-HRP, Jiaxuan Bio.) was added and incubated at 37°C for 30 minutes; after 5-fold washing with PBS, 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-5 min; then 100 µl of 2N H ₂ SO ₄ stopping solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the blocking effect of SS002M91, SG3847L1, SG3847L2 on CD47/SIRPα was analyzed.

As seen in Figure 10, similarly to SS002M91, SG3847L1 and SG3847L2 can competitively block binding between CD47 and its SIRPα ligand. The IC ₅₀ value for SG3847L1 is 46.8 nM, the IC ₅₀ value for SG3847L2 is 78.8 nM, and the IC ₅₀ value for SS002M91 is 45.7 nM.

Embodiment 2: Use of Combinations of Different Target Antibodies and Human SIRP α Variant 1 Mutant

According to the structure of the fusion protein shown in Figure 1 and the results of Embodiment 1, taking anti-AXL antibody (C6G12) and anti-Trop2 antibody (SG701) for example, the effect of various target antibodies on the biological activity of the fusion protein in which as linker used linker 2.

The amino acid sequences of LCDR1-3 of the C6G12 antibody are shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 28; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 29; the amino acid sequence of the light chain is shown in SEQ ID NO: 32; and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 34.

The amino acid sequences of the LCDR1-3 antibody SG701 are shown in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively; the amino acid sequence of VL is shown in SEQ ID NO: 42; the amino acid sequences of HCDR1-3 are shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively; the amino acid sequence of VH is shown in SEQ ID NO: 43; the amino acid sequence of the light chain is shown in SEQ ID NO: 46; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 48.

The fusion protein was prepared from an anti-AXL antibody (C6G12), an M91 variant 1 SIRPα mutant, and linker 2 (SEQ ID: NO 74). C6G12, a linker and two M91s were sequentially linked from N-terminus to C-terminus, with the N-terminus of two M91s linked to the C-terminus of the C6G12 heavy chain, respectively, and the resulting fusion protein was named SGAXL47. The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGAXL47 are shown in SEQ ID NO: 77 and SEQ ID NO: 32, respectively.

The fusion protein was prepared from an anti-Trop antibody (SG701), an M91 variant 1 SIRPα mutant, and linker 2 (SEQ ID: NO 74). SG701, a linker and two M91s were sequentially linked from N-terminus to C-terminus, with the N-terminus of two M91s linked to the C-terminus of the SG701 heavy chain, respectively, and the resulting fusion protein was named SGTrop247. The amino acid sequences of the second polypeptide chain and the first polypeptide chain of SGTrop247 are shown in SEQ ID NO: 78 and SEQ ID NO: 46, respectively.

Using standard molecular biology techniques, the SGAXL47 and SGTrop247 fusion protein expression genes were inserted into eukaryotic expression vectors. After transfection of CHO-S cells, the supernatant was collected, and then affinity purification was carried out through protein A to obtain the target protein, the biological activity of which was evaluated accordingly.

2.1 Two antigen binding activity assay

(1) Using humanized antibodies against various antigens as a control, the binding activity between the fusion protein and related antigens was evaluated by ELISA.

Target antigens were applied to ELISA plates, incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; various concentrations of antibodies or fusion proteins were added and reacted at 37° C. for 1 hour; after washing with PBS, horseradish peroxidase-labeled secondary goat anti-human IgG (Goat Anti human IgG HRP, Thermo Fisher Scientific) was added, reacted at 37°C for 30 minutes, and washed with PBS 5 times; 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-2 minutes; then 100 µl of 2N H ₂ SO ₄ stopping solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the ability of the fusion protein to bind the respective target antigens was analyzed.

As shown in Figure 11A, the ability of SGAXL47 to bind to the AXL antigen is close to that of C6G12. As shown in Figure 11B, the ability of SGTrop247 to bind to the Trop2 antigen is close to that of SG701. This demonstrated that antibodies with different target sites used in the fusion protein did not affect the ability of the antibody to bind to the Trop2 antigen.

(2) Using SS002M91 as a control, the binding activity between the fusion protein and CD47 was assessed by ELISA.

The target CD47 antigen was applied to ELISA plates, incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; various concentrations of SS002M91 fusion protein or fusion protein were added and reacted at 37° C. for 1 hour; after washing with PBS, horseradish peroxidase-labeled secondary goat anti-human IgG (Goat Anti human IgG HRP, Thermo Fisher Scientific) was added, reacted at 37°C for 30 minutes, and washed with PBS 5 times; 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-2 minutes; then 100 μl of 2N H ₂ SO ₄ stop solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the CD47 binding ability of the fusion protein was analyzed.

As shown in Figure 12A, the ability of SGAXL47 to bind to the CD47 antigen is close to that of SS002M91. As shown in Figure 12B, the ability of SGTrop247 to bind to the CD47 antigen is close to that of SS002M91. This demonstrated that antibodies with different target sites used in the fusion protein did not affect the ability of the antibody to bind the CD47 antigen.

2.2. Analysis of the activity of simultaneous binding of two antigens

Using humanized antibodies against various antigens as a control, the biological activity of the fusion protein to bind two antigens simultaneously was analyzed by solid phase ELISA.

applied to ELISA plates, incubated overnight at 4°C; after washing with PBST, 10% fetal calf serum was added and blocked at 37°C for 1 hour; various concentrations of antibodies or fusion proteins were added and reacted at 37° C. for 1 hour; after washing with PBS, biotin-labeled CD47 (Biotin-Fc-CD47) was added, the reaction was carried out at a temperature of 37°C for 30 minutes, and PBS was washed 5 times; avidin labeled with horseradish peroxidase (Streptavidin-HRP, Jiaxuan Bio.) was added, reacted at 37°C for 30 minutes, and washed with PBS 5 times; 100 µl of TMB (eBioscience) was added to each well, placed in the dark at room temperature (20±5°C) for 1-2 minutes; then 100 µl of 2N H ₂ SO ₄ stopping solution was added to each well to stop the substrate reaction, OD values were read at 450 nm on a microplate reader, and the ability of the fusion proteins to simultaneously bind the respective antigen and CD47 was analyzed.

Figure 13A shows the binding profiles of SGAXL47 and C6G12 to the dual antigens AXL and CD47, respectively; Figure 13B shows the binding profiles of SGTrop247 and SS002M91 to the Trop2 and CD47 dual antigens, respectively. The results shown in Figure 13 showed that different antibodies substituted in the fusion protein did not affect its simultaneous binding to dual antigens.

Embodiment 3 Fusion Protein Inhibits Tumor Activity in vivo

CB17 mice with SCID syndrome were inoculated with luciferase-expressing Raji cells by intravenous tail injection to create tumor models, and the inhibitory effect of the SG3847L1 fusion protein on tumor activity was evaluated.

5-8 week old female CB17 SCID mice (purchased from Beijing Biocytogen Co., Ltd) were selected for the experiment. Luciferase-expressing Raja cells were stable cell lines based on Raja cells obtained by introducing a luciferase reporter gene, these cells were purchased from Beijing Biocytogen Co., Ltd. Luciferase-expressing Raja cells were reconstituted and cultured to the desired number, then the cells were harvested during the logarithmic growth phase and suspended to a concentration of 2.5 x 10 ⁷ per ml. This suspension was intravenously administered to CB17 Mice with SCID by tail injection at a dose of 0.2 ml per mouse. On days 0 and 7 after administration, a small animal thermal imager was used to monitor growth profile and tumor weight. On day 7, 30 mice with moderate tumor imaging signals were selected and these mice were randomly assigned to 5 groups, 6 mice per group. The animals were then given single injections of drugs that were divided into a saline control group, an experimental group (SG003, 100 µg/kg), an experimental group (SS002M91, 50 µg/kg), a combined dose group (SG003+SS002M91, 100 µg/kg+50 mcg/kg), experimental group (SG3847L1, 120 mcg/kg). At the same time, the weight of mice and tumor growth profiles were monitored.

The results showed that the SG003 antibody, the SS002M91 fusion protein and the SG3847L1 fusion protein were tolerated by the experimental animals. Compared with the control group treated with saline, there was a clear inhibitory effect on tumor growth in different groups of animals that were dosed (see Figure 14). Figure 15 shows that on day 7 after administration, the mean fluorescence intensity of the SG003 antibody treatment group, the SS002M91 fusion protein treatment group, the SG003 antibody and SS002M91 fusion protein combination treatment group, and the SG3847L1 fusion protein treatment group was 1.60E+07 (standard error: 1.02E+07), 2.42E+07 (standard error: 6.70E+06), 4.61E+06 (standard error: 7.57E+05), 5.97E+05 (standard error: 1, 10E+05), respectively, all of these values were significantly lower than the mean fluorescence intensity of the control group, ie 8.04E+08 (standard error: 1.15E+08). In addition, the inhibitory effect on tumor growth in the SG3847L1 fusion protein treatment group was significantly superior to that in the group treated with the combination of the SG003 antibody and the SS002M91 fusion protein.

In addition, in Figure 16, "saline G1", "G2 SG003", "G3 SS002M91", "G4 SG003+SS002M91", and "G5 SG3847L1" denote "saline solution group", "SG003 antibody treatment group", "treatment group SS002M91 fusion protein", "SG003 and SS002M91 combination treatment group", and "SG3847L1 fusion protein treatment group", respectively; The median survival time in the saline treated group was 11 days, the median survival time in the SG003 antibody treatment group and the SS002M91 fusion protein treatment group was 22 days and 21 days, respectively, indicating that SG003 and SS002M91 significantly extended survival time mice. Up to 23 days after grouping, there were no deaths in the SG003 and SS002M91 combination treatment group and in the SG3847L1 fusion protein treatment group, indicating that, compared with treatment with SG003 or SS002M91 alone, the survival time of mice in the SG003 and SS002M91 combination treatment groups SS002M91 and in the treatment group only SG3847L1 was extended.

The above detailed description is provided by way of explanation and examples, but is not intended to limit the scope of the appended claims. The various embodiments of the invention described above in this application are obvious to those of ordinary skill in the art and are within the scope of the appended claims and their equivalents.

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SEQUENCE LIST

<110> Hangzhou Sumgen Biotech Co., Ltd.

Sumgen Mab (Beijing) Biotech Co., Ltd.

<120> FUNCTION PROTEIN AND ITS USE

<130> 0070-PA-014

<160> 78

<170> Patent Version 3.5

<210> 1

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR1 SG003

<400> 1

Arg Ala Ser Ser Ser Val Ser Ser Ser Ala Phe Ser Tyr Val His

1 5 10 15

<210> 2

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR2 SG003

<400> 2

Leu Ala Ser Asn Leu Glu Ser

15

<210> 3

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR3 SG003

<400> 3

His His Ser Arg Glu Leu Pro Phe Thr

15

<210> 4

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR1 SG003

<400> 4

Leu Tyr Trp Met Asn

15

<210> 5

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR2 SG003

<400> 5

Lys Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu Lys

1 5 10 15

asp

<210> 6

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR3 SG003

<400> 6

Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr

1 5 10

<210> 7

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> VL SG003

<400> 7

Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Ala Phe Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 8

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> VH SG003

<400> 8

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 9

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding VL SG003

<400> 9

gagatcgtga tgacccagag ccctgccagc ctgagcgcca gcctgggcca gagggccacc 60

atcagctgca gggccagcag cagcgtgagc agcagcgcct tcagctacgt gcactggtac 120

cagcagaaga gcggccagcc tcctaagctg ctgatctacc tggccagcaa cctggagagc 180

ggcgtgcctg ccaggttcag cggcagcggc agcggcaccg acttcaccct gaccatccac 240

cctgtggaga gcgaggacgt ggccacctac tactgccacc acagcaggga gctgcctttc 300

accttcggca gcggcaccaa gctggagatc aag 333

<210> 10

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding VH SG003

<400> 10

caggtgcagc tgctggagag cggcggcggc ctggtgcagc ctggcggcag cctgaagctg 60

agctgcgtgg ccagcggctt cgacttcagc ctgtactgga tgaactgggt gaggcaggcc 120

cctggcaagg gcctggagtg gatcggcaag atcaaccctg acagcagcac catcaactac 180

acccctagcc tgaaggacaa gttcttcatc agcagggaca acgccaagaa caccctgtac 240

ctgcagatga ccaaggtgag gagcgaggac accgccctgt actactgcgc caggctgtgg 300

atcgccaccg gcggcttcga ctactggggc cagggcacca ccctgaccgt gagcagc 357

<210> 11

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain SG003

<400> 11

Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Ala Phe Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Leu Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 12

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding light chain SG003

<400> 12

gagatcgtga tgacccagag ccctgccagc ctgagcgcca gcctgggcca gagggccacc 60

atcagctgca gggccagcag cagcgtgagc agcagcgcct tcagctacgt gcactggtac 120

cagcagaaga gcggccagcc tcctaagctg ctgatctacc tggccagcaa cctggagagc 180

ggcgtgcctg ccaggttcag cggcagcggc agcggcaccg acttcaccct gaccatccac 240

cctgtggaga gcgaggacgt ggccacctac tactgccacc acagcaggga gctgcctttc 300

accttcggca gcggcaccaa gctggagatc aagcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aaggcttca acaggggaga gtgttag 657

<210> 13

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain SG003

<400> 13

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 14

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding heavy chain SG003

<400> 14

caggtgcagc tgctggagag cggcggcggc ctggtgcagc ctggcggcag cctgaagctg 60

agctgcgtgg ccagcggctt cgacttcagc ctgtactgga tgaactgggt gaggcaggcc 120

cctggcaagg gcctggagtg gatcggcaag atcaaccctg acagcagcac catcaactac 180

acccctagcc tgaaggacaa gttcttcatc agcagggaca acgccaagaa caccctgtac 240

ctgcagatga ccaaggtgag gagcgaggac accgccctgt actactgcgc caggctgtgg 300

atcgccaccg gcggcttcga ctactggggc cagggcacca ccctgaccgt gagcagcgct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacgtgcg tggtggtgga cgtgagccac gaagaccccg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

cagggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaatga 1350

<210> 15

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> Binding epitope of CD38 protein

<400> 15

Ser Thr Phe Gly Ser Val Glu Val His Asn Leu Gln Pro Glu Lys Val

1 5 10 15

Gln Thr Leu Glu Ala Trp

20

<210> 16

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant light chain 1 SG003

<400> 16

Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gln Arg Ala Ser Ile Ser Cys Arg Ala Ser Asn Ser Val Ser Ser Ser

20 25 30

Ala Tyr Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Ile Gln Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Gln Leu Pro Ser Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant heavy chain 1 SG003

<400> 17

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Tyr Asn Phe Ser Leu Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Gln Pro Glu Ser Ser Thr Ile Gln Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Trp Ile Gly Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 18

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant light chain 2 SG003

<400> 18

Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser

20 25 30

Ala Tyr Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asp Leu Gln Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Leu Pro Tyr Ser Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 19

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant heavy chain 2 SG003

<400> 19

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Ser Pro Asn Ser Ser Thr Ile Asn Tyr Ser Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Tyr Ile Ala Ser Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 20

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant light chain 3 SG003

<400> 20

Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Asn Ser Val Ser Thr Ser

20 25 30

Ala Phe Ser Tyr Val His Trp Tyr Gln Gln Lys Ser Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Ser Glu Asp Val Ala Thr Tyr Tyr Cys His His Ser Arg

85 90 95

Glu Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Mutant heavy chain 3 SG003

<400> 21

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Ser Pro Asp Ser Ser Ser Leu Asn Tyr Thr Pro Ser Val

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Tyr Asn Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 22

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR1 C6G12

<400> 22

Arg Ser Ser Arg Ser Leu Leu His Ser Asn Gly Phe Thr Tyr Leu Tyr

1 5 10 15

<210> 23

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR2 C6G12

<400> 23

Gln Met Ser Asn Leu Ala Ser

15

<210> 24

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR3 C6G12

<400> 24

Gly Gln Asn Leu Glu Leu Pro Leu Thr

15

<210> 25

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR1 C6G12

<400> 25

Ser Gly Tyr Tyr Trp Asn

15

<210> 26

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR2 C6G12

<400> 26

Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn

1 5 10 15

<210> 27

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR3 C6G12

<400> 27

Gly Trp Leu Leu His Tyr Thr Met Asp Tyr

1 5 10

<210> 28

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> VL C6G12

<400> 28

Glu Leu Val Met Thr Gln Ser Pro Phe Ser Asn Ala Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser

20 25 30

Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn

85 90 95

Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 29

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> VH C6G12

<400> 29

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 30

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding VL C6G12

<400> 30

gagctcgtga tgacacagtc tccattctcc aatgcagtca ctcttggaac atcagcttcc 60

atctcctgca ggtctagtag gagtctccta catagtaatg gcttcactta tttgtattgg 120

tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180

tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240

agcagagtgg aggctgagga tgtgggtgtt tattactgtg gtcaaaatct agagcttccg 300

ctcacgttcg gtgctgggac caagctggag ctgaaa 336

<210> 31

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding VH C6G12

<400> 31

caggtgcagc tgaagcagtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60

acctgttctg tcactggctt ctccatcagc agtggttatt actggaactg gatccggcag 120

tttccaggaa acaaactgga atggatgggc tacagaagct acgacggttc caataactac 180

aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa ccagtttttc 240

ctgaagttga attctgtgac aactgaggac acagctacat attactgtgc aagaggatgg 300

ttactgcatt atactatgga ctactggggt caaggaacct cagtcaccgt ctcctca 357

<210> 32

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain C6G12

<400> 32

Glu Leu Val Met Thr Gln Ser Pro Phe Ser Asn Ala Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu His Ser

20 25 30

Asn Gly Phe Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gln Asn

85 90 95

Leu Glu Leu Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 33

<211> 660

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding light chain C6G12

<400> 33

gagctcgtga tgacacagtc tccattctcc aatgcagtca ctcttggaac atcagcttcc 60

atctcctgca ggtctagtag gagtctccta catagtaatg gcttcactta tttgtattgg 120

tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc 180

tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240

agcagagtgg aggctgagga tgtgggtgtt tattactgtg gtcaaaatct agagcttccg 300

ctcacgttcg gtgctgggac caagctggag ctgaaacgta cggtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540

agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600

660

<210> 34

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain C6G12

<400> 34

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 35

<211> 1350

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding heavy chain C6G12

<400> 35

caggtgcagc tgaagcagtc aggacctggc ctcgtgaaac cttctcagtc tctgtctctc 60

acctgttctg tcactggctt ctccatcagc agtggttatt actggaactg gatccggcag 120

tttccaggaa acaaactgga atggatgggc tacagaagct acgacggttc caataactac 180

aacccatctc tcaaaaatcg aatctccatc actcgtgaca catctaagaa ccagtttttc 240

ctgaagttga attctgtgac aactgaggac acagctacat attactgtgc aagaggatgg 300

ttactgcatt atactatgga ctactggggt caaggaacct cagtcaccgt ctcctcagct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacgtgcg tggtggtgga cgtgagccac gaagaccccg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

cagggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaatga 1350

<210> 36

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR1 SG701

<400> 36

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 37

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR2 SG701

<400> 37

Asp Ala Ser Asn Leu Glu Ala

15

<210> 38

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> LCDR3 SG701

<400> 38

Gln Gln Tyr Gly Asp Phe Pro Leu Thr

15

<210> 39

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR1 SG701

<400> 39

Asn Asn Tyr Ile His

15

<210> 40

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR2 SG701

<400> 40

Met Ile Asn Pro Ser Gly Gly Ser Thr Thr Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 41

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> HCDR3 SG701

<400> 41

Ala Ala Leu Ser Tyr Tyr Ser Ile Val Thr Ala Lys Asp Tyr Gly Met

1 5 10 15

Asp Val

<210> 42

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> VL SG701

<400> 42

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Ala Gly Val Pro Ser Arg Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Gly Asp Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 43

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> VH SG701

<400> 43

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Asn

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asn Pro Ser Gly Ser Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Leu Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Leu Ser Tyr Tyr Ser Ile Val Thr Ala Lys Asp Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 44

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding VL SG701

<400> 44

gacatccagc tgacccagag ccctagcagc gtgagcgcca gcgtgggcga cagggtgaac 60

atcacctgca gggccagcca gggcatcagc agctggctgg cctggtacca gcagaagcct 120

ggcaaggccc ctaagctgct gatctacgac gccagcaacc tggaggccgg cgtgcctagc 180

aggttcaggg gcagcggcag cggcaccgac ttcaccttca ccatcaccag cctgcagcct 240

gaggacatcg ccacctactt ctgccagcag tacggcgact tccctctgac cttcggccag 300

ggcaccaagc tggagatcaa g 321

<210> 45

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding VH SG701

<400> 45

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ctggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc aacaactaca tccactgggt gaggcaggcc 120

cctggccagg gcctggagtg gatgggcatg atcaacccta gcggcggcag caccacctac 180

240

atggacctga gcagcctgag gagcgaggac accgccgtgt actactgcgc cagggccgcc 300

ctgagctact acagcatcgt gaccgccaag gactacggca tggacgtgtg gggccaggggc 360

acccgtga ccgtgagcag c 381

<210> 46

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain SG701

<400> 46

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Asn Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Ala Gly Val Pro Ser Arg Phe Arg Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Thr Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Gly Asp Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 47

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding SG701 light chain

<400> 47

gacatccagc tgacccagag ccctagcagc gtgagcgcca gcgtgggcga cagggtgaac 60

atcacctgca gggccagcca gggcatcagc agctggctgg cctggtacca gcagaagcct 120

ggcaaggccc ctaagctgct gatctacgac gccagcaacc tggaggccgg cgtgcctagc 180

aggttcaggg gcagcggcag cggcaccgac ttcaccttca ccatcaccag cctgcagcct 240

gaggacatcg ccacctactt ctgccagcag tacggcgact tccctctgac cttcggccag 300

ggcaccaagc tggagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645

<210> 48

<211> 457

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain SG701

<400> 48

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Asn

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asn Pro Ser Gly Ser Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Leu Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Leu Ser Tyr Tyr Ser Ile Val Thr Ala Lys Asp Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120 125

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

130 135 140

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

145 150 155 160

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

165 170 175

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

180 185 190

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

195 200 205

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

210 215 220

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

225 230 235 240

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

245 250 255

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

260 265 270

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

275 280 285

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

325 330 335

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

340 345 350

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

355 360 365

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

370 375 380

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

385 390 395 400

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

405 410 415

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

420 425 430

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 49

<211> 1374

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid encoding SG701 heavy chain

<400> 49

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ctggcgccag cgtgaaggtg 60

agctgcaagg ccagcggcta caccttcacc aacaactaca tccactgggt gaggcaggcc 120

cctggccagg gcctggagtg gatgggcatg atcaacccta gcggcggcag caccacctac 180

240

atggacctga gcagcctgag gagcgaggac accgccgtgt actactgcgc cagggccgcc 300

ctgagctact acagcatcgt gaccgccaag gactacggca tggacgtgtg gggccaggggc 360

acccgtga ccgtgagcag cgctagcacc aagggcccat cggtcttccc cctggcaccc 420

tcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480

cccgaaccgg tgacggtgtc gtggaactca ggcgccctga ccagcggcgt gcacaccttc 540

ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600

agcagcttgg gcaccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660

gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 720

gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 780

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccacgaagac 840

cccgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900

ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 960

caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1020

cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1080

ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140

ggcttctatc ccagcgacat cgccgtggag tgggagca atgggcagcc ggagaacaac 1200

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260

accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320

gctctgcaca accactacac gcagaagagc ctctccctgt ctcggggtaa atga 1374

<210> 50

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Human SIRPa variant 1 truncated domain

<400> 50

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> 51

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M1

<400> 51

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 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 Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 52

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M5

<400> 52

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 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> 53

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M12

<400> 53

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 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 Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 54

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M35

<400> 54

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 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 Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 55

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M37

<400> 55

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 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> 56

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M41

<400> 56

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 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> 57

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M57

<400> 57

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 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 Arg Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 58

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M67

<400> 58

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 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 Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 59

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M81

<400> 59

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 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> 60

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M82

<400> 60

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 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 Ser Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 61

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M84

<400> 61

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 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 Ser Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 62

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M91

<400> 62

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 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 Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 63

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M99

<400> 63

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 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 Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 64

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M102

<400> 64

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 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 Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 65

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M111

<400> 65

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 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 Leu Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser

115

<210> 66

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M122

<400> 66

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 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> 67

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M126

<400> 67

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 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> 68

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M130

<400> 68

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 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> 69

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M135

<400> 69

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 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> 70

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> M145

<400> 70

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 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> 71

<211> 233

<212> Protein

<213> Artificial sequence

<220>

<223> IgG1-FC

<400> 71

Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 72

<211> 350

<212> Protein

<213> Artificial sequence

<220>

<223> SS002M91

<400> 72

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 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 Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

100 105 110

Arg Ala Lys Pro Ser Leu Glu Pro Lys Ser Cys Asp Lys Thr His Thr

115 120 125

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

130 135 140

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

145 150 155 160

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

165 170 175

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

180 185 190

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

195 200 205

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

210 215 220

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

225 230 235 240

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

245 250 255

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

260 265 270

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

275 280 285

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

290 295 300

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

305 310 315 320

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

325 330 335

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

340 345 350

<210> 73

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Linker 1

<400> 73

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 74

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Linker 2

<400> 74

Gly Gly Gly Ser Ala Ala Ala

15

<210> 75

<211> 576

<212> Protein

<213> Artificial sequence

<220>

<223> Second polypeptide chain SG3847L1

<400> 75

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Val Ile

450 455 460

Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu

465 470 475 480

Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly Pro Ile Gln Trp Phe

485 490 495

Arg Gly Ala Gly Pro Gly Arg Gln Leu Ile Tyr Asn Gln Lys Asp Gly

500 505 510

His Phe Pro Arg Val Thr Thr Ala Ser Asp Leu Thr Lys Arg Asn Asn

515 520 525

Met Asp Phe Ser Ile Asn Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly

530 535 540

Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ile Glu

545 550 555 560

Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser

565 570 575

<210> 76

<211> 573

<212> Protein

<213> Artificial sequence

<220>

<223> Second polypeptide chain SG3847L2

<400> 76

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Phe Asp Phe Ser Leu Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu

50 55 60

Lys Asp Lys Phe Phe Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Thr Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Leu Trp Ile Ala Thr Gly Gly Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys Gly Gly Gly Ser Ala Ala Ala Glu Leu Gln Val Ile Gln Pro Asp

450 455 460

Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr

465 470 475 480

Ala Thr Ser Leu Leu Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala

485 490 495

Gly Pro Gly Arg Gln Leu Ile Tyr Asn Gln Lys Asp Gly His Phe Pro

500 505 510

Arg Val Thr Thr Ala Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe

515 520 525

Ser Ile Asn Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr

530 535 540

Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ile Glu Phe Lys Ser

545 550 555 560

Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser

565 570

<210> 77

<211> 573

<212> Protein

<213> Artificial sequence

<220>

<223> Second polypeptide chain SGAXL47

<400> 77

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Phe Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Arg Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp Leu Leu His Tyr Thr Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys Gly Gly Gly Ser Ala Ala Ala Glu Leu Gln Val Ile Gln Pro Asp

450 455 460

Lys Ser Val Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr

465 470 475 480

Ala Thr Ser Leu Leu Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala

485 490 495

Gly Pro Gly Arg Gln Leu Ile Tyr Asn Gln Lys Asp Gly His Phe Pro

500 505 510

Arg Val Thr Thr Ala Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe

515 520 525

Ser Ile Asn Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr

530 535 540

Cys Val Lys Phe Arg Lys Gly Ser Pro Asp Asp Ile Glu Phe Lys Ser

545 550 555 560

Gly Ala Gly Thr Glu Leu Ser Val Arg Ala Lys Pro Ser

565 570

<210> 78

<211> 581

<212> Protein

<213> Artificial sequence

<220>

<223> Second polypeptide chain SGTrop247

<400> 78

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Asn

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile Asn Pro Ser Gly Ser Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Leu Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Ala Leu Ser Tyr Tyr Ser Ile Val Thr Ala Lys Asp Tyr

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120 125

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

130 135 140

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

145 150 155 160

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

165 170 175

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

180 185 190

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

195 200 205

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

210 215 220

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

225 230 235 240

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

245 250 255

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

260 265 270

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

275 280 285

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

325 330 335

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

340 345 350

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Asp Glu Leu

355 360 365

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

370 375 380

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

385 390 395 400

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

405 410 415

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

420 425 430

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Ser Ala Ala Ala

450 455 460

Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly

465 470 475 480

Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Leu Pro Val Gly

485 490 495

Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Gln Leu Ile Tyr

500 505 510

Asn Gln Lys Asp Gly His Phe Pro Arg Val Thr Thr Ala Ser Asp Leu

515 520 525

Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Asn Ile Gly Asn Ile Thr

530 535 540

Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser

545 550 555 560

Pro Asp Asp Ile Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val

565 570 575

Arg Ala Lys Pro Ser

580

<---

Claims (44)

1. A fusion protein for binding to a CD47 protein and a tumor-associated antigen, comprising: a first binding domain that specifically binds the tumor-associated antigen; and a second binding domain that specifically binds the CD47 protein; where said second binding domain contains a human SIRPα domain mutant, wherein said human SIRPα domain contains amino acid residues at positions 33-149 of human SIRPα variant 1, where said amino acid residues at positions 33-149 of human SIRPα variant 1 are shown in SEQ ID NO: 50, and said mutant contains amino acid substitutions selected from the group consisting of: (1) I61L, V63I, E77I, E84K, V93L, L96S, K98R, N100G and V132L; (2) I61V, E77N, Q82S, K83R and E84H; (3) I61F, V63I, K83R, E84K and V132I; (4) I61L, E77Q, E84D, R107N and V132I; (5) I61L, V63I, E77K, K83R, E84D and N100G; (6) I61V, E77H, Q82R, K83R, E84H and R107S; (7) I61L, E77I, Q82G, E84R, V93L, L96T, N100G, R107S, G109R and V132R; (8) I61L, E77M, Q82G, K83R, E84D and V132L; (9) I61L; (10) I61F, D95H, L96S, G109H and V132S; (11) I61F, D95H, L96S, K98R, G109H and V132S; (12) I61L, E77Q, E84D, V93A, R107N and V132I; (13) E77K, L96S, N100K, G109H and V132L; (14) I61L, V63I, Q82G, E84G, D95R, L96S, N100D and V132I; (15) I61L, E77R, Q82N, K83R, E84G, V93L, D95E, L96T, K98R, N100D and V132L; (16) I61V, E77N, Q82S, K83R, E84H and V93A; (17) I61V, V63I, E77V, K83R, E84D, D95E, L96T, K98R and N100E; (18) I61L, V63I, E77V, K83R, D95E, L96S, K98R, N100D and G109R; (19) I61V, E77L, Q82G, E84G, V93L, D95E, L96T, K98R and N100G; And (20) I61L, V63I, E77N, Q82G and E84G. 2. A fusion protein according to claim 1, wherein said mutant contains the amino acid sequence shown in any of SEQ ID NOs: 51-70. 3. The fusion protein of claim 1, wherein said first binding domain comprises an antibody or an antigen-binding fragment or variant thereof. 4. The fusion protein of claim 1, wherein said tumor-associated antigen is selected from the group consisting of CD38, AXL, and Trop2. 5. The fusion protein of claim 1, wherein the first binding domain comprises an anti-CD38 antibody, or an antigen-binding fragment or variant thereof. 6. The fusion protein according to claim 5, wherein said antibody contains the heavy chain of the antibody or fragment thereof, wherein the heavy chain of the antibody or fragment thereof contains HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, in sequence. 7. The fusion protein according to claims 5, 6, where the antibody contains the light chain of the antibody or its fragment, where the light chain of the antibody or its fragment contains LCDR1-3, the amino acid sequences of LCDR1-3 are SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, in sequence. 8. The fusion protein of claim 4, wherein the first binding domain comprises an anti-AXL antibody or an antigen-binding fragment or variant thereof. 9. The fusion protein of claim 8, wherein the antibody comprises a heavy chain of an antibody or fragment thereof, wherein the heavy chain of the antibody or fragment thereof comprises HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, consecutively. 10. A fusion protein according to any one of claims 8, 9, wherein the antibody comprises a light chain of an antibody or fragment thereof, wherein the light chain of the antibody or fragment thereof contains LCDR1-3, the amino acid sequences of LCDR1-3 are SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, in sequence. 11. The fusion protein of claim 4, wherein the first binding domain comprises an anti-Trop2 antibody, or an antigen-binding fragment or variant thereof. 12. The fusion protein of claim 11 wherein the antibody comprises a heavy chain of the antibody or fragment thereof, wherein the heavy chain of the antibody or fragment thereof comprises HCDR1-3, the amino acid sequences of HCDR1-3 are SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, sequentially. 13. Fusion protein according to any one of paragraphs. 11, 12, where the antibody contains the light chain of the antibody or fragment thereof, where the light chain of the antibody or fragment thereof contains LCDR1-3, the amino acid sequences of LCDR1-3 are SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38 , sequentially. 14. The fusion protein of claim 1, wherein the first binding domain is located at the N-terminus of the second binding domain. 15. The fusion protein of claim 1, wherein the fusion protein further comprises a linker, wherein the linker is located at the C-terminus of the first binding domain and is located at the N-terminus of the second binding domain. 16. A fusion protein according to claim 1, comprising at least two of the second binding domains, and each of the second binding domains is located at the C-terminus of the first binding domain, respectively. 17. A fusion protein according to claim 1, containing a first polypeptide chain and a second polypeptide chain, where the first polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 11 and the second polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 75, the first polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 11 and the second polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 76, the first polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 46 and the second polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 78, or the first polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 32 and the second polypeptide chain contains the amino acid sequences shown in SEQ ID NO: 77. 18. Composition for binding to the CD47 protein and/or tumor-associated antigen, containing an effective amount of the fusion protein according to any one of paragraphs. 1-17 and optionally a pharmaceutically acceptable excipient. 19. The use of a fusion protein according to any one of paragraphs. 1-17 or a composition according to claim 18 for the preparation of a medicament, wherein the medicament is used to treat a tumor, wherein said tumor includes multiple myeloma, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, neuroglioma, germinoma, sarcoma, mesothelioma, placentomas, brain cancer, bone cancer, skin cancer, nasopharyngeal cancer, lung cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, ovarian cancer and testicular cancer. 20. The use of a fusion protein according to any one of paragraphs. 1-17 or a composition according to claim 18 for the preparation of a medicament, wherein the medicament is used to treat an autoimmune disease, wherein said autoimmune disease includes chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma and polyarteritis nodosa.