Fusion protein and application thereof

Abstract

A fusion protein composed of SIRP [alpha] V2 and a CD3 antibody is provided.

Description

Fusion proteins and their applications

[0001] This application claims priority from Chinese patent application No. CN202211163564.8, filed on September 23, 2022. The description, drawings, and claims of this priority document are incorporated herein in their entirety and made a part of the original description of this application. The applicant further declares that the applicant reserves the right to amend the description and claims of this application based on this priority document. Technical Field

[0002] The present application relates to the field of biomedicine, and specifically to a fusion protein consisting of SIRPαV2 and CD3 antibody. Background Art

[0003] Fusion proteins can simultaneously recognize two different targets, influencing the initiation of more cellular activation regulatory mechanisms, and have enormous potential in the treatment of tumors and other diseases. T cell engager bispecific antibodies are a typical example of this type of drug. Their mechanism of action is to simultaneously bind to T cell surface antigens (such as CD3) and tumor cell surface antigens (such as CD19) to form an immune synapse (as shown in Figure 1), thereby shortening the distance between tumor cells and T cells and directly activating and proliferating T cells. These T cells can then be used to directly kill tumor cells or release cytotoxins to kill them. Generally speaking, this type of T cell activation process is simple and direct, and does not require the presentation of tumor cell antigens to generate specific T lymphocyte clones. Therefore, it is not restricted by MHC or HLA and has a high clinical conversion rate.

[0004] CD47 is a transmembrane protein ubiquitously present on the surface of normal cells and belongs to the immunoglobulin superfamily. It is an important target for the next generation of immuno-oncology therapies. CD47 and PD-1, respectively, target two major immune cell populations that play a major role in cancer immunotherapy. The CD47 signaling pathway primarily regulates macrophages, while the PD-1 pathway primarily regulates T lymphocytes. Macrophages are a crucial component of innate immunity and function as scavengers in the human body by phagocytosing senescent and dying cells. CD47 is highly expressed on various solid tumors and hematological malignancies, and its expression level positively correlates with disease progression. Because it inhibits macrophage phagocytosis of tumor cells after binding to SIRPα, CD47 is often overexpressed on the surface of tumor cells, aiding them in immune evasion. Theoretically, CD47 pathway drugs, like PD-1 drugs, could be used to treat a wide range of cancer types. Currently, CD47-targeted drugs, both monotherapy and in combination, are being used in the targeted treatment of common hematological and solid tumors, including leukemia, lymphoma, lung cancer, and liver cancer.

[0005] Summary of the Invention

[0006] There are three known natural ligands for CD47: integrins, platelet aggregation protein-1, and SIRPα. Its biological roles include cell adhesion, cell migration, regulation of inflammatory responses, and inhibition of macrophage phagocytosis. Given the aforementioned immune checkpoint functions of CD47 and SIRPα, the use of the SIRPαV domain in molecular design can help target CD47. By preventing CD47 on the surface of cancer cells from binding to SIRPα on the surface of macrophages or dendritic cells, it can relieve the inhibitory effect of cancer cells on macrophages, induce macrophage phagocytosis of tumor cells, promote the uptake of tumor cells by dendritic cells, and facilitate the presentation of tumor antigens.

[0007] Since CD47 is also expressed on the surface of normal cells (especially red blood cells) and the SIRPαV2 SNP variant exhibits selective weak affinity for CD47 on the surface of red blood cells, in order to avoid off-target toxicity, the selection of the SIRPαV2 variant in molecular design can greatly alleviate problems such as cell aggregation and lysis, as well as thrombocytopenia.

[0008] This application combines SIRPαV2 with CD3 antibodies to form a fusion protein, thereby activating T cells and bringing tumor cells and T cells closer to induce killing of tumor cells. At the same time, SIRPa can also activate the inhibitory effect of contacting tumor cells on macrophages and DCs, further weakening the immune escape of tumor cells. When designing this fusion protein, we compared the symmetrical configuration (D1-TW) and the asymmetric configuration (H3S02). It was found that the asymmetric configuration was significantly higher than the symmetrical configuration in terms of efficacy, and the release levels of several different cytokines were close to or lower than those of the symmetrical configuration molecules. In addition, this application also provides an asymmetric molecule Mos-D1-TW, which uses a different anti-CD3 sequence, but the other parts are the same as D1-TW.

[0009] In one aspect, the application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion;

[0010] wherein the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the heavy chain variable region HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is as shown in any one of SEQ ID NOs: 4, 24, 32 and 40; the amino acid sequence of the HCDR2 is as shown in any one of SEQ ID NOs: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is as shown in any one of SEQ ID NOs: 6, 26, 34 and 42, and / or the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the light chain variable region LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is as shown in any one of SEQ ID NOs: 1, 27, 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NOs: 2, 28, 36 and 44; and the amino acid sequence of the LCDR3 is as shown in any one of SEQ ID NOs: 3, 29, 37 and 45, wherein the CD3 binding portion can induce T cell activation.

[0011] In certain embodiments, the CD47 binding portion comprises an anti-CD47 antibody or an antigen-binding fragment thereof, or a CD47 ligand or a functional variant thereof.

[0012] In certain embodiments, the CD47 ligand comprises integrin, thrombohemagglutinin-1, SIRPγ, SIRPα, or functional variants or fragments thereof.

[0013] In certain embodiments, the CD47 binding portion comprises SIRPα, or a functional variant thereof, or a fragment thereof.

[0014] In another aspect, the present application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion;

[0015] wherein the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the amino acid sequence of HCDR1 is as shown in any one of SEQ ID NOs: 4, 24, 32, and 40; the amino acid sequence of HCDR2 is as shown in any one of SEQ ID NOs: 5, 25, 33, and 41; and the amino acid sequence of HCDR3 is as shown in any one of SEQ ID NOs: 6, 26, 34, and 42, and / or the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the light chain variable regions LCDR1, LCDR2, and LCDR3, and the amino acid sequence of LCDR1 is as shown in any one of SEQ ID NOs: 1, 27, 35, and 43; the amino acid sequence of LCDR2 is as shown in any one of SEQ ID NOs: 2, 28, 36, and 44; and the amino acid sequence of LCDR3 is as shown in any one of SEQ ID NOs: 3, 29, 37, and 45, wherein the CD3 binding portion is capable of inducing T cell activation;

[0016] The CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the SIRPα protein, and the amino acid sequence of the SIRPα protein is shown in SEQ ID NO: 46.

[0017] In another aspect, the present application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion that specifically binds to human CD47 and human CD3, wherein the CD3 binding portion competes with a reference antigen binding protein for binding to human CD3, the reference antigen binding protein comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3; and

[0018] The CD47 binding portion competes with the SIRPα protein for binding to human CD47, and the SIRPα protein comprises the amino acid sequence shown in SEQ ID NO:46.

[0019] In another aspect, the present application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion, wherein the CD3 binding portion comprises three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 4, 24, 32, and 40; wherein HCDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 5, 25, 33, and 41; wherein HCDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 6, 26, 34, and 42, wherein LCDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 27, 35, and 43, wherein LCDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 2, 28, 36, and 44, and wherein LCDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 3, 29, 37, and 45; and

[0020] The CD47 binding portion comprises the amino acid sequence shown in SEQ ID NO:46.

[0021] In certain embodiments, the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3; wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:3;

[0022] wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:24; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:25; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:26, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:27, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:28 (DTS), and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:29;

[0023] wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:32; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:33; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:34, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:35, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:36, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:37; or

[0024] wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:44 (YTS), and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:45.

[0025] In certain embodiments, the CD3 binding portion comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 8, 15, 16, 22, 30 and 38; and / or the CD3 binding portion comprises a light chain variable region, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 7, 19, 23, 31 and 39.

[0026] In certain embodiments, the CD3 binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7;

[0027] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;

[0028] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;

[0029] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23;

[0030] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; or

[0031] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39.

[0032] In certain embodiments, the CD3 binding moiety is an antibody or an antigen-binding fragment thereof.

[0033] In certain embodiments, the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0034] In certain embodiments, the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0035] In certain embodiments, the fusion protein further comprises a heterodimeric Fc portion.

[0036] In certain embodiments, the Fc is derived from the Fc of IgG1, IgG2, IgG3, or IgG4.

[0037] In certain embodiments, the heterodimeric Fc portion is connected by a disulfide bond in the hinge region and a knob-in-hole structure in the CH3 domain.

[0038] In certain embodiments, the CD3 binding portion comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3 or IgG4.

[0039] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17.

[0040] In certain embodiments, the CD3 binding portion comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

[0041] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:20.

[0042] In certain embodiments, the CD3 binding portion comprises an antibody heavy chain and a light chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 21.

[0043] In certain embodiments, the CD47 binding portion comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3 or IgG4.

[0044] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:47.

[0045] In certain embodiments, the CD47 binding portion comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:48.

[0046] On the other hand, the present application provides a fusion protein having two heavy chains and one light chain, wherein the first heavy chain has VH-CH1-hinge region-Fc from N-terminus to C-terminus, and the first light chain has VL-CL from N-terminus to C-terminus; the second heavy chain has SIRPα-hinge region-Fc from N-terminus to C-terminus, wherein the VH-CH1 of the first heavy chain and the VL-CL of the first light chain form an antigen binding site that binds to CD3, and the SIRPα of the second heavy chain binds to CD47;

[0047] wherein the first heavy chain comprises three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3), the first light chain comprises three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 4, 33, 41 and 49; wherein HCDR2 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 5, 34, 42 and 50; wherein HCDR3 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 6, 35, 43 and 51, wherein LCDR1 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 1, 36, 44 and 52, wherein LCDR2 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 2, 37, 45 and 53, and wherein LCDR3 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 3, 38, 46 and 54; and wherein the second heavy chain comprises SIRPα, wherein the SIRPα comprises the amino acid sequence as shown in SEQ ID NO: 46.

[0048] In certain embodiments, the first heavy chain comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the first light chain comprises light chain variable regions LCDR1, LCDR2, and LCDR3; wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:3;

[0049] wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29;

[0050] wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:32; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:33; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:34, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:35, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:36, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:37; or

[0051] wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:44, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:45.

[0052] In certain embodiments, the first heavy chain comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NOs: 8, 15, 16, 22, 30 and 38; and / or the first light chain comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 7, 19, 23, 31 and 39.

[0053] In certain embodiments, the first heavy chain comprises a heavy chain variable region, and the first light chain comprises a light chain variable region; wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 7;

[0054] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;

[0055] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19;

[0056] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23;

[0057] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; or

[0058] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39.

[0059] In certain embodiments, the first heavy chain and the second heavy chain comprise Fc from IgG.

[0060] In certain embodiments, the Fc is an Fc from IgG1, IgG2, IgG3, or IgG4.

[0061] In certain embodiments, the first heavy chain and the second heavy chain comprise Fc from human IgG.

[0062] In certain embodiments, the Fc is an Fc from human IgG1, human IgG2, human IgG3, or human IgG4.

[0063] In certain embodiments, the first heavy chain and the second heavy chain are connected via a disulfide bond in the hinge region and a knob-in-hole structure in the CH3 domain.

[0064] In certain embodiments, wherein the first heavy chain and the second heavy chain are of the human IgG1 isotype, and wherein one of the first heavy chain or the second heavy chain comprises T366S, L368A, and Y407V heavy chain substitutions, and the other of the first heavy chain or the second heavy chain comprises a T366W heavy chain substitution, wherein the residues are numbered according to the EU index.

[0065] In certain embodiments, the first light chain comprises a CL from a human lambda or kappa light chain.

[0066] In certain embodiments, the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain comprises the amino acid sequence shown in SEQ ID NO: 21.

[0067] In certain embodiments, the second heavy chain comprises the amino acid sequence shown in SEQ ID NO:48.

[0068] On the other hand, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein described in the present application or the fusion protein described in the present application.

[0069] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0070] In the present application, nucleic acids encoding the antibodies and antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific procedures, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0071] In another aspect, the present application provides a vector comprising a nucleic acid described herein. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Furthermore, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence functionally linked to the nucleic acid for transcriptional control. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a certain portion of the promoter may be fused to a certain portion of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene promoters, which may or may not include other introns. One or more nucleic acid molecules described herein may be operably connected to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0072] On the other hand, the application provides a kind of cell, it includes according to the carrier described in the present application.In certain embodiments, every kind or each host cell comprises one or a kind of nucleic acid molecule or carrier described in the present application.In certain embodiments, every kind or each host cell comprises multiple (for example, 2 or more) or multiple (for example, 2 kinds or more) nucleic acid molecules or carrier described in the present application.For example, the carrier described in the present application can be introduced into the host cell, for example eukaryotic cell, such as from plant cell, fungus or yeast cell etc.The carrier described in the present application can be introduced into the host cell by methods known in the art, for example electroporation, lipofectine transfection, lipofectamin transfection etc.

[0073] On the other hand, the present application provides a pharmaceutical composition comprising the fusion protein described herein and a pharmaceutically acceptable carrier.

[0074] The pharmaceutical composition of the present application may contain a safe and effective amount (such as 0.001-99wt%, 0.01-90wt%, or 0.1-80wt%) of the fusion protein described in the present application and a pharmaceutically acceptable carrier. Such carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition described in the present application can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the fusion protein described in the present application can also be used in conjunction with other therapeutic agents.

[0075] Fusion protein or pharmaceutical composition as described herein can be prepared, administered and used in a manner consistent with good medical practice. Considerations in this case include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of disease, the agent delivery site, the method of administration and other factors known to medical practitioners. The therapeutic agent need not but is optionally prepared and / or administered simultaneously with one or more medicaments currently used to prevent or treat the disease under consideration. The effective amount of such other agents depends on the amount of the therapeutic agent present in the preparation, the type of disease or treatment and other factors discussed above. These agents can usually be determined as any dosage that is appropriate empirically / clinically and are used by any approach that is determined as appropriate empirically / clinically. Compared with single treatment, the dosage of the antibody used in the combination therapy can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.

[0076] On the other hand, the present application provides the use of the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein in the preparation of a drug for treating cancer.

[0077] In certain embodiments, the cancer comprises solid tumors and hematological tumors.

[0078] In certain embodiments, the cancer is a CD47-expressing cancer.

[0079] In certain embodiments, the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.

[0080] On the other hand, the present application provides a method for treating cancer in a subject, comprising administering to the subject the fusion protein described herein, the isolated nucleic acid molecule described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein, thereby inhibiting the growth of the cancer in the subject.

[0081] In certain embodiments, the cancer comprises solid tumors and hematological tumors.

[0082] In certain embodiments, the cancer is a CD47-expressing cancer.

[0083] In certain embodiments, the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.

[0084] In certain embodiments, it further comprises administering a second therapeutic agent to the subject.

[0085] In certain embodiments, the second therapeutic agent is an anti-tumor agent, radiation therapy, an antibody drug conjugate, a checkpoint inhibitor, or a combination thereof.

[0086] On the other hand, the present application provides a method for producing the fusion protein described in the present application or the fusion protein described in the present application, wherein the method comprises culturing the host cell described in the present application under conditions capable of expressing the fusion protein.

[0087] In certain embodiments, the host cell is selected from bacterial cells, fungal cells, plant cells, mammalian cells or viruses.

[0088] In certain embodiments, the bacterial cell is Escherichia coli.

[0089] In certain embodiments, the fungal cell is a yeast cell.

[0090] In certain embodiments, the mammalian cells are selected from CHO, NS0, BHK or HEK293 cells.

[0091] In certain embodiments, the cell is a hybridoma cell.

[0092] In certain embodiments, the hybridoma cells are selected from mouse, rat, or rabbit.

[0093] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0095] FIG1 shows the configurations of the asymmetric anti-CD3×SIRPa-Fc Fusion Protein (D1-TW) and the symmetric anti-CD3×SIRPa (H3S02) described in this application.

[0096] Figure 2 shows a map of the vector p2MPT.

[0097] Figure 3 shows a map of the vector pMPTN.

[0098] Figure 4 shows the two-step purification diagram of the Mos-D1-TW molecule.

[0099] FIG5 shows the SDS-PAGE image of Mos-D1-TW after molecular purification.

[0100] Figure 6 shows the two-step purification of the D1-TW molecule.

[0101] FIG7 shows the SDS-PAGE image of D1-TW after molecular purification.

[0102] FIG8 shows a diagram of H3S02 molecular purification.

[0103] FIG9 shows the SDS-PAGE image of H3SO2 molecules after purification.

[0104] FIG10 shows the results of affinity testing of D1-TW to CD3E / G and CD47.

[0105] FIG11 shows the ELISA test results of Mos-D1-TW dual antibody.

[0106] FIG12 shows the binding of D1-TW to 8226 and HCT-8 tumor cells detected by FACS.

[0107] FIG13 shows the binding of mos-D1-TW to target cells 8226 and PBMC of hCD3E transgenic mice detected by FACS.

[0108] FIG14 shows the killing effect of hPBMC on tumor cells 8226 and HCT-8 mediated by D1-TW.

[0109] Figures 15-16 show the release of cytokines during D1-TW-mediated killing of two tumor cell lines, 8226 and HCT-8. DETAILED DESCRIPTION

[0110] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0111] Definition of terms

[0112] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0113] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this article, the scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form, unless otherwise clearly provided in the text.

[0114] In this application, the term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0115] In the present application, the term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0116] SIRPα is a protein belonging to the SIRP receptor family. The full Chinese name of SIRP is signal regulatory proteins (SIRP). It is mainly expressed on the surface of myeloid cells (monocytes, macrophages, granulocytes, and myeloid DC cells, etc.), and is also expressed in neuronal cells of the nervous system. Structurally, SIRPα contains three extracellular immunoglobulin superfamily domains, including an N-terminal variable region (V region) that binds to CD47 and two C1-type immunoglobulin superfamily domains, which are then connected to the intracellular inhibitory signaling domain through a transmembrane helical domain. In this application, the term "CD3" generally refers to a part of the T cell receptor complex, consisting of three different chains CD3ε, CD3δ and CD3γ. The concentration of CD3 on T cells, such as by the fixation of anti-CD3 antibodies, leads to the activation of T cells, which is similar to T cell receptor-mediated activation, but does not depend on the specificity of the TCR clone. The vast majority of anti-CD3 antibodies recognize the CD3ε chain. The term refers to any native CD3 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed CD3 as well as any form of CD3 produced by intracellular processing, or any fragment thereof. The term also includes naturally occurring variants of CD3, e.g., splice variants or allelic variants. In a preferred embodiment, CD3 refers to full-length CD3 from humans and cynomolgus monkeys, or fragments thereof (such as mature fragments lacking a signal peptide). In a preferred embodiment, CD3 refers to full-length CD3 from mice / rat, or fragments thereof (such as mature fragments lacking a signal peptide).

[0117] In this application, the term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed to determine percent amino acid sequence identity using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0118] In this application, the term "functional variant" generally refers to a nucleic acid molecule or protein comprising a nucleotide and / or amino acid sequence that changes one or more nucleotides and / or amino acids compared to the nucleotide and / or amino acid sequence of a reference nucleic acid molecule or binding molecule. In other words, the modification of the amino acid and / or nucleotide sequence of the reference binding molecule does not significantly affect or change the binding properties of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the binding molecule is still able to recognize and bind its target. The functional variants of a gene include gene variants with minor changes, for example, silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly change gene function. Functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and random PCR-mediated mutagenesis), and may include natural and non-natural nucleotides and amino acids.

[0119] In this application, the term "immune response" generally refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and soluble macromolecules (including antibodies, cytokines and complement) produced by the above cells or the liver, which results in the selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0120] In this application, the term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0121] In this application, the terms "activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CD47 activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS or BIACORE plasmon resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0122] In this application, the term "fusion protein" generally refers to a polypeptide or protein that contains the amino acid sequence of a first polypeptide or protein, or a fragment, analog or derivative thereof, and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein, or a fragment, analog or derivative thereof, that is different from the first polypeptide or protein, or a fragment, analog or derivative thereof, or that is not generally a part of the first polypeptide or protein, or a fragment, analog or derivative thereof). In some cases, a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide. The heterologous protein, polypeptide or peptide may or may not be a different type of prophylactic or therapeutic drug. For example, two different proteins, polypeptides or peptides having immunomodulatory activity can be fused together to form a fusion protein. In some cases, the fusion protein retains or increases the activity compared to the activity of the original polypeptide or protein before fusion with the heterologous protein, polypeptide or protein.

[0123] In the present application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb), immunoconjugates, multispecific antibodies (e.g., fusion proteins), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they show the desired antigen binding activity. The "isolated antigen binding protein" of the present application may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the binding of the antigen binding portion to the antigen.

[0124] In this application, the term "antibody" generally refers to any form of antibody with the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as fusion proteins), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies. It is known that the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical polypeptide chain pairs, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The amino-terminal portion or fragment of each chain may include a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion or fragment of each chain may define a constant region that is primarily responsible for effector function. Human light chains are generally classified as kappa and lambda light chains. In addition, human heavy chains are generally classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)).

[0125] In this application, the term "isolated antibody" generally refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0126] In this application, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method.

[0127] In this application, the term "fusion protein" generally refers to an artificially designed antibody that is composed of components of two different antigen binding sites and can bind to two different antigen binding sites at the same time.

[0128] In this application, the term "full-length antibody" generally refers to an immunoglobulin molecule that, when naturally present, comprises four peptide chains: two heavy (H) chains (approximately 50-70 kDa when full-length) and two light (L) chains (approximately 25 kDa when full-length), interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0129] In this application, the term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0130] As used herein, the term "single-chain Fv" or "scFv" antibody generally refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0131] In this application, the term "complementarity determining region (CDR)" is the region of an antibody that binds to an antigen. CDRs can be defined using various descriptors, such as Kabat (Wu et al., 1970, J Exp Med, Vol. 132, pp. 211-250) (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., 1987, J Mol Biol, Vol. 196, pp. 901-917), IMGT (Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77), and AbM (Martin and Thornton, 1996, J Bmol Biol, Vol. 263, pp. 800-815). The correspondence between various depictions and variable region numbers is described (see, for example, Lefranc et al., 2003, Dev Comp Immunol, Vol. 27, pp. 55-77; Honegger and Pluckthun, 2001, J Mol Biol, Vol. 309, pp. 657-670; International Immunogenetics (IMGT) database; Web resources, http: / / www_imgt_org). Available programs (such as abYsis of UCL Business PLC) can be used to depict CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT or AbM). For example, the CDR regions of the present application can be defined using the Kabat rules.

[0132] As used herein, the term "Fc," "Fc region," or "Fc fragment" generally refers to a polypeptide comprised of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM, connected by a hinge region. While the breakdown of the Fc fragment varies, the heavy chain Fc fragment of human IgG generally refers to the polypeptide extending from A231 to its carboxyl terminus.

[0133] In this application, the term "hinge region" generally refers to the proline-rich, easily stretchable and bendable polypeptide chain located between CH1 and CH2 in an antibody. The generally accepted IgG hinge region is the polypeptide chain consisting of amino acid residues 216 to 230.

[0134] In this application, the term "domain antibody" generally refers to an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0135] In this application, the term "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0136] In this application, the term "diabody" generally refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0137] In this application, the term "chimeric antibody" generally refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody ("parent antibody") such as a rodent, while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0138] In the present application, the term "humanized antibody" generally refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that is engineered to contain minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin, wherein the residues from the complementary determining region (CDR) are replaced by residues from the CDRs of non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and ability (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In some cases, the Fv framework region (FW) residues of a human immunoglobulin are replaced by corresponding residues from antibodies of non-human species with desired specificity, affinity, and ability.

[0139] In this application, the term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0140] In this application, an antibody "isotype" generally refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modification has been made to alter Fc function, for example to enhance or diminish effector function or binding to Fc receptors.

[0141] In this application, the term "epitope" generally refers to the region of an antigen to which an antibody binds. An epitope can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0142] In this application, "affinity" or "binding affinity" generally refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0143] In this application, the term "not binding" to a protein or cell generally means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., the KD of the binding protein or cell is 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0×10 -2 M or higher.

[0144] In this application, the term "high affinity" for IgG antibodies generally refers to a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10-9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0145] As used herein, the terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively lyse target cells, such as cancer cells, that have antibody-bound cell surface antigens.

[0146] In this application, the term "complement-dependent cytotoxicity" or "CDC" generally refers to the effector functions of IgG and IgM antibodies, which, when bound to surface antigens, trigger the classical complement pathway, including formation of the membrane attack complex and target cell lysis.

[0147] In this application, the term "nucleic acid" or "polynucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids having similar binding properties to reference nucleic acids and analogs containing known natural nucleotides that are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0148] In this application, "construct" generally refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with the genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner. Recombinant constructs will generally comprise a polynucleotide of the present invention operably linked to transcription initiation regulatory sequences that direct transcription of the polynucleotide in the host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0149] In this application, "vector" generally refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0150] In this application, the term "expression vector" generally refers to a nucleic acid molecule that is capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0151] In this application, "activation", "stimulation" and "treatment" as used for cells or receptors may have the same meaning, e.g., a cell or receptor is activated, stimulated or treated with a ligand, unless the context otherwise or clearly dictates. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response / reaction", e.g., the response of a cell, tissue, organ or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate or differentiation state), where the change is related to activation, stimulation or treatment, or to internal mechanisms such as genetic programming.

[0152] In this application, the " treatment " of term any disease or illness refers to improving disease or illness (that is, slowing down or preventing or reducing at least one of the progress of disease or its clinical symptoms) in one embodiment. In another embodiment, " treatment " refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, " treatment " refers to physically (for example, the stabilization of discernible symptoms), physiologically (for example, the stabilization of physical parameters) or regulating disease or illness in these two aspects. Unless clearly described in this article, the method for the treatment and / or prevention of the assessment of disease is generally known in the art.

[0153] In this application, "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.

[0154] In the present application, administration "in combination with" one or more other therapeutic agents includes simultaneous (concurrent) administration and consecutive administration in either order.

[0155] As used herein, "therapeutically effective amount," "therapeutically effective dose," and "effective amount" generally refer to an amount of an antigen binding protein of the invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount to treat, cure, prevent, or ameliorate the relevant medical condition or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40%, and most preferably by at least 50%.

[0156] As used herein, the terms "cancer" and "cancerous" generally refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included within this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, storage diseases (bulky disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0157] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0158] Example

[0159] 1. Antibody Expression

[0160] Sequences encoding the asymmetric Linton anti-CD3 antibody heavy chain and light chain were inserted into the p2MPT vector into the EcoRI / XbaI expression cassette and the NotI / BamHI expression cassette, respectively, to construct recombinant plasmid 1, p2MPT-hDL-D1TW-GA. Sequences encoding the SIRPa-Fc Fusion protein were ligated into the p2MPT vector into the NotI / BamHI expression cassette to construct recombinant plasmid 2, p2MPT-hS1-GA. After verification by sequencing and enzyme digestion, the correctly constructed plasmids 1 and 2 were prepared using the QIAGEN Plasmid Midi Kit.

[0161] The sequences encoding the heavy and light chains of the competitor anti-CD3 antibody were ligated into the NotI / BamHI expression cassettes of the pMPTN vector to construct recombinant plasmids 3pMPTN-MD1TW-HC and 4pMPTN-MD1TW-LC, respectively. After verification by gene sequencing and enzyme digestion, the correctly constructed plasmids 3 and 4 were prepared using the Endo-Free Plasmid Maxi Kit (Omega).

[0162] The sequences encoding the symmetrical heavy and light chains of the anti-CD3 x SIRPa antibody were ligated into the NotI / BamHI expression cassette and the EcoRI / XbaI expression cassette, respectively, of the p2MPT vector to construct recombinant plasmid 5, p2MPT-H3S02-HC-C3002LC. After verification by sequencing and enzyme digestion, the correctly constructed plasmid 5 was prepared using the QIAGEN Plasmid Midi Kit.

[0163] For expression of asymmetric Linton anti-CD3 x SIRPa-Fc Fusion Protein (D1-TW) and symmetric Linton anti-CD3 x SIRPa (H3S02), CHO-DG44-express cells stored in liquid nitrogen were revived and cultured (Ex-cell Advanced CHO Fed-Batch Medium, Sigma) at 37°C, 180 rpm, 75% RH, and 5% CO2. Transfection and expression were performed after cell viability stabilized to above 98%. The cell density at the time of transfection was 3.0×10 6cells / ml, using proCHO5 medium (Lonza) and 25K polyethyleneimine (PEI, Sigma) as the transfection reagent. DNA was used at a ratio of 3 μg / mL (10% of the total cell culture volume, with the helper plasmid pBase used) to PEI at a 1:3 ratio. Linton anti-CD3 plasmid and SIRPa-Fc Fusion plasmid were used at a 1:1 ratio. 24 hours after transfection, the medium was changed to Ex-cell Advanced CHO Fed-Batch Medium (Sigma), and selection was performed using puromycin (Enzo). During selection, the cell density was set at 5 x 10^5 cells / mL, and the final puromycin concentration was 10 μg / mL. Antibody expression was performed when cell viability stabilized above 98%. After 7 days of expression at 31°C, 180 rpm, 75% RH, and 5% CO2, the supernatant was collected by centrifugation.

[0164] For expression of the asymmetric Competitor anti-CD3 x SIRPa-Fc Fusion Protein, Expi293F (Gibco) cells stored in liquid nitrogen were revived and cultured (Wayne 293 Transfection Medium, Kang Tianshenghe). Culture conditions were 37°C, 110 rpm, 75% RH, and 6% CO2. Transfection was performed after cell viability stabilized to above 98%. The cell density at the time of transfection was 1.5×10 6 cells / ml, using 25K polyethyleneimine (PEI, Sigma) as the transfection reagent. DNA was used at a concentration of 1 μg / mL (cell culture volume) in a 1:4 ratio of PEI. Competitor anti-CD3 heavy and light chain plasmids and SIRPa-Fc Fusion plasmids were used in a 1:1:1 ratio. Six days after transfection, the supernatant was collected by centrifugation.

[0165] 2. Antibody enrichment

[0166] The CHO cell supernatant was centrifuged at 2000 rpm to remove cells and 8000 rpm to remove cell debris. The clarified culture medium was filtered through a 0.45 μm microporous filter. After equilibration of the Mabselect Sure column with PBS, the antibodies with Fc termini in the CHO cell supernatant were fully enriched at a retention time of 4 min / min. The column was re-equilibrated with PBS and eluted with 20 mM sodium citrate buffer (pH 3.0). The sample was collected and adjusted to pH 5.6 with Tris-HCl (pH 8.5).

[0167] 3. Double antibody purity

[0168] 3.1 Ion chromatography

[0169] The Mos-D1TW molecule was purified using cation chromatography. An SPHP column was equilibrated with 20 mM sodium citrate buffer (pH 5.4), and the enriched antibody solution was then passed through an SP HP column at 1 ml / min. After injection, the column was equilibrated with 20 mM sodium citrate buffer (pH 5.4). Finally, a linear elution was performed from A: 20 mM sodium citrate buffer (pH 5.4) to 35% B: 20 ​​mM sodium citrate buffer, 1 M NaCl (pH 5.4), over 20 CV. Samples were collected separately to yield Peak 1 (approximately 110 kDa) and Peak 2 (impurity).

[0170] 3.2 Hydrophobic chromatography

[0171] The D1-TW molecule was purified using a hydrophobic column. A UniHR Phenyl-30L column was equilibrated with 50mM sodium phosphate, 1M ammonium sulfate (pH 6.8) buffer. The enriched antibody solution was then added to 2M ammonium sulfate (pH 6.8) in a 1:1 ratio, mixed, and passed through the UniHR Phenyl-30L column at 1 ml / min. After injection, the column was equilibrated again with 50mM sodium phosphate, 1M ammonium sulfate (pH 6.8). Finally, a linear elution was performed from A: 50mM sodium phosphate, 1M ammonium sulfate (pH 6.8) to 100% B: 50mM sodium phosphate buffer over 20 CV. Samples were collected separately to yield Peak 1 (approximately 120 kDa) and Peak 2 (regenerated).

[0172] 4. In vitro functional activity analysis

[0173] 4.1 Elisa assay for D1-TW activity

[0174] CD47-hFc (Yi Qiao, 12283-H02H) 1.5ug / ml was coated with 0.05M CB solution and incubated at 4°C overnight; the next day, the plate was washed three times with PBST and blocked with 1xPBS + 1% BSA Elisa buffer, 200ul per well, and placed in a 37°C incubator for 1 hour; the sample D1-TW to be tested was prepared by diluting D1-TW with Elisa buffer, starting from 9ug / ml and performing a 3-fold gradient dilution, with a total of 10 concentration points including point 0; the blocked Elisa test plate was removed from the incubator, patted dry, and the diluted antibody was added, 100ul per well, and placed in a 37°C incubator for 1 hour; the test plate was removed, washed three times with PBST, and human CD3E&G-hFc-his (Nearshore, C08G) was diluted to 2ug / ml with Elisa buffer, 100ul per well, and placed in a 37°C incubator for 1h; the detection plate was removed, washed three times with PBST, and anti-his-HRP (Gensher, A00612, 1:1K) was added, 100ul per well, and placed in a 37°C incubator for 1h; the detection plate was removed, washed three times with PBST, and TMB color development solution was added, 100ul per well, and color development was performed for 10min; 1M H2SO4 was added, 50ul per well, to terminate the reaction, and OD450 was read on a microplate reader.

[0175] Results: As shown in Figure 10, D1-TW has the affinity to bind to both CD3E / G and CD47.

[0176] 4.2 Elisa validation of Mos-D1-TW dual antibody

[0177] hCD3E-his (C578) was coated with 0.05M CB solution at 1ug / ml at 4°C overnight; the next day, the plate was washed three times with PBST, blocked with 1xPBS+1% BSA Elisa buffer, 200ul per well, and placed in a 37°C incubator for 1h; the sample Mos-D1-TW to be tested was prepared by diluting D1-TW with Elisa buffer, starting from 10ug / ml, with a 10-fold gradient dilution, including 0 point for a total of 5 concentration points; the blocked Elisa test plate was removed from the incubator, patted dry, and the diluted antibody was added at 100ul per well, and placed in a 37°C incubator for 1h; the test plate was removed, washed three times with PBST, and anti-SIRPa-mFc (abcam, ab267409) was added, diluted to 1ug / ml with Elisa buffer, 100ul per well, and placed in a 37°C incubator for 1h; the test plate was removed, washed three times with PBST, and Goat anti-mouse Fc-HRP (Jacksonimmuno, 115-035-071, 1:5K), 100ul per well, placed in a 37°C incubator for 1h; remove the test plate, wash three times with PBST, add TMB color development solution, 100ul per well, and develop for 10min; add 1M H2SO4, 50ul per well, stop, and read OD450 on a microplate reader.

[0178] As shown in FIG11 , the expressed and purified mos-D-TW is a complete and effective bispecific antibody.

[0179] 4.3 FACS detection of D1-TW binding to target cells 8226 and HCT-8

[0180] D1-TW was diluted in 3% BSA FACS buffer to a starting concentration of 50 μg / ml and serially diluted 3X to 9 concentrations, including 0 μg / ml. HCT-8 tumor cells were trypsinized, and 8226 cells were collected and centrifuged at 200g for 5 minutes. The cells were resuspended in 3% BSA, counted, and the cell density adjusted to 2E6 cells / ml. 100 μl of diluted antibody was added to each well of a 96-well U-shaped plate and centrifuged at 200g for 5 minutes. The supernatant was removed. The plate was then added with 100 μl of 1xPBS and resuspended. A negative control well without antibody was also set up. The plate was reacted at 4°C for 30 minutes, centrifuged at 200g for 5 minutes, the supernatant removed, and the plate was washed once with 3% BSA. The fluorescent secondary antibody FITC mouse anti-human IgG Fc was added and reacted at 4°C for 30 minutes. The plate was centrifuged at 200g for 5 minutes, the supernatant removed, and the plate was washed once with 1xPBS. The plate was centrifuged at 200g for 5 minutes, and the supernatant removed. The plate was resuspended in 100 μl / well of 1xPBS and analyzed by flow cytometry.

[0181] The results are shown in Figure 12. D1-TW maintains the ability to bind to both 8226 and HCT-8 tumor cell lines.

[0182] 4.4 FACS detection of mos-D1-TW binding to target cells 8226 and PBMCs of hCD3E transgenic mice

[0183] D1-TW was diluted with 3% BSA FACS buffer to a starting concentration of 50 μg / ml, and 5X serial dilutions were performed, including 0 μg / ml for a total of 6 concentration points. 8226 cells were collected, centrifuged at 200g for 5 minutes, resuspended in 3% BSA, counted, and the cell density adjusted to 2E6 cells / ml. 100 μl was added to each well of a 96-well U-shaped plate, centrifuged at 200g for 5 minutes, and the supernatant was removed. Meanwhile, whole blood from hCD3E transgenic mice was collected and lysed twice with erythrocyte lysis buffer for 10 minutes each. The cells were washed twice with 1xPBS, resuspended, and 100 μl was added to each well. The cells were centrifuged at 200g for 5 minutes, and the supernatant was removed. The diluted antibody was added to 100 μl per well and resuspended. A negative control well without antibody was set up, reacted at 4°C for 30 minutes, centrifuged at 200g for 5 minutes, the supernatant was removed, and the wells were washed once with 3% BSA. The fluorescent secondary antibody FITC mouse anti-human IgG was added. Fc, react at 4 degrees for 30 minutes, centrifuge at 200g for 5 minutes, remove the supernatant, wash once with 1xPBS, centrifuge at 200g for 5 minutes, remove the supernatant; resuspend with 100ul / well 1xPBS, and detect by flow cytometry.

[0184] The results are shown in FIG13 , showing that Mos-D1-TW has a certain affinity for target cells 8226 and CD3E on the cell surface.

[0185] 4.5 hPBMCs’ killing effect on D1-TW-mediated tumor cell 8226 and HCT-8

[0186] (1) PBMC preparation:

[0187] The frozen PBMCs were revived, counted, and the cell density was adjusted to 2×10^6 cells / ml. 50 μl / well was added to a 96-well U-shaped plate, i.e., 1×10^5 cells / well.

[0188] (2) Preparation of tumor cells 8226 and HCT-8DE:

[0189] Take a T75 cell culture flask of normally cultured tumor cells, digest HCT-8 with 2 ml of trypsin 0.25% EDTA, resuspend in 8 ml of cell culture medium, centrifuge at 1000 rpm for 5 minutes, remove the supernatant, wash the cells once with 5 ml of culture medium, remove the supernatant, resuspend in 2 ml of culture medium, count the cells, adjust the cell density to 1 × 10^5 cells / ml, and add 100 μl / well to a 96-well plate containing PBMCs, i.e., 1 × 10^4 cells / well, and pre-adhere to the wall and culture for 2 hours; at the same time, take 10 ml of 8226 cells with a pipette, stain with FAR-red-APC, count the cells, adjust the cell density to 1 × 10^5 cells / ml, and add 100 μl / well to a 96-well plate containing PBMCs;

[0190] (3) Antibody preparation:

[0191] Prepare D1-TW in culture medium with a starting concentration of 40,000 ng / ml and perform a 5-fold serial dilution. 50 μl of each concentration of antibody solution was dispensed into a 96-well plate. The final concentration was 10,000 ng / ml. Perform a 5-fold serial dilution, including 0 ng / ml, for a total of 11 concentration points. Single wells for PBMC and tumor cells were also set up.

[0192] (4) Place in a carbon dioxide incubator (37°C, 5% CO2) and culture for 24 h, 48 h, 72 h, and 96 h respectively;

[0193] (5) For CCK8: After culturing in a carbon dioxide incubator (37°C, 5% CO2) for 24h, 48h, 72h, and 96h, the culture supernatant was aspirated for cytokine kit detection; the cultured cells were washed twice with PBS to remove PBMC, and then 100μl / well of CCK8 working solution was added. After incubation at 37°C for 2-4 hours, OD450 was measured; and for the 8226 culture system, 1ul of PI staining reagent was added to each well, protected from light for 10min, and then flow cytometry was performed.

[0194] The killing results are shown in Figure 14. D1-TW can mediate efficient killing of two tumor cell lines, 8226 and HCT-8, and is more effective than the symmetrical structure H3S02.

[0195] 4.6 Cytokine Detection

[0196] The culture supernatant of the above culture system after killing for 48 hours was obtained and cytokine detection kits Human IL-2 Uncoated ELISA Kit (Invitrogen, 88-7025-88), IL-6 Human Uncoated ELISA Kit (Invitrogen, 88-7066-77), Human IFN-γ ELISA Kit (Biolegend, 430107), TNF alpha Human Uncoated ELISA Kit (eBioscience, 88-7346-76), and Human Granzyme B ELISA BASIC kit (HRP) (mabtech, 3486-1H-20) were used according to the kit instructions. The detection results are shown in Figures 15-16:

[0197] Results: During the D1-TW-mediated killing of 8226 and HCT-8 tumor cell lines, the cytokines released were similar to or lower than those of the symmetrical structure H3S02.

Claims

1. An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion; wherein the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the heavy chain variable region HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is as shown in any one of SEQ ID NOs: 4, 24, 32 and 40; the amino acid sequence of the HCDR2 is as shown in any one of SEQ ID NOs: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is as shown in any one of SEQ ID NOs: 6, 26, 34 and 42, and / or the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the light chain variable region LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is as shown in any one of SEQ ID NOs: 1, 27, 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NOs: 2, 28, 36 and 44; and the amino acid sequence of the LCDR3 is as shown in any one of SEQ ID NOs: 3, 29, 37 and 45, wherein the CD3 binding portion can induce T cell activation.

2. The fusion protein according to claim 1, wherein the CD47 binding portion comprises an anti-CD47 antibody or an antigen-binding fragment thereof, or a CD47 ligand or a functional variant thereof. 3 . The fusion protein according to claim 2 , wherein the CD47 ligand comprises integrin, thrombohemagglutinin-1, SIRPγ, SIRPα or functional variants or fragments thereof. 4 . The fusion protein according to claim 1 , wherein the CD47 binding portion comprises SIRPα, or a functional variant thereof, or a fragment thereof.

5. An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion; wherein the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the amino acid sequence of HCDR1 is as shown in any one of SEQ ID NOs: 4, 24, 32, and 40; the amino acid sequence of HCDR2 is as shown in any one of SEQ ID NOs: 5, 25, 33, and 41; and the amino acid sequence of HCDR3 is as shown in any one of SEQ ID NOs: 6, 26, 34, and 42, and / or the CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the light chain variable regions LCDR1, LCDR2, and LCDR3, and the amino acid sequence of LCDR1 is as shown in any one of SEQ ID NOs: 1, 27, 35, and 43; the amino acid sequence of LCDR2 is as shown in any one of SEQ ID NOs: 2, 28, 36, and 44; and the amino acid sequence of LCDR3 is as shown in any one of SEQ ID NOs: 3, 29, 37, and 45, wherein the CD3 binding portion is capable of inducing T cell activation; The CD3 binding portion comprises an amino acid sequence that is at least 95% identical to the SIRPα protein, and the amino acid sequence of the SIRPα protein is shown in SEQ ID NO:

46.

6. An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion that specifically binds to human CD47 and human CD3, wherein the CD3 binding portion competes for binding to human CD3 with a reference antigen binding protein comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:4; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:6, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3; and The CD47 binding portion competes with the SIRPα protein for binding to human CD47, and the SIRPα protein comprises the amino acid sequence shown in SEQ ID NO:

46.

7. An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion, wherein the CD3 binding portion comprises three heavy chain complementary determining regions (HCDR1, HCDR2, and HCDR3) and three light chain complementary determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 4, 24, 32, and 40; wherein HCDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 5, 25, 33, and 41; wherein HCDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 6, 26, 34, and 42, wherein LCDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 1, 27, 35, and 43, wherein LCDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 2, 28, 36, and 44, and wherein LCDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 3, 29, 37, and 45; and The CD47 binding portion comprises the amino acid sequence shown in SEQ ID NO:

46.

8. The fusion protein according to any one of claims 1 to 7, wherein the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3 and light chain variable regions LCDR1, LCDR2 and LCDR3; wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:3; or; wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29; or; wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 35, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37; Or; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:44, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

45.

9. The fusion protein according to any one of claims 1 to 8, wherein the CD3 binding portion comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 8, 15, 16, 22, 30 and 38; and / or the CD3 binding portion comprises a light chain variable region, and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NOs: 7, 19, 23, 31 and 39.

10. The fusion protein according to any one of claims 1 to 9, wherein the CD3 binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

39. The fusion protein according to any one of claims 1 to 10, wherein the CD3 binding portion is an antibody or an antigen-binding fragment thereof. The fusion protein according to claim 11 , wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.

13. The fusion protein of any one of claims 11-12, wherein the antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

14. The fusion protein according to any one of claims 1 to 13, further comprising a heterodimeric Fc portion. The fusion protein according to claim 14 , wherein the Fc is derived from the Fc of IgG1, IgG2, IgG3 or IgG4. The fusion protein according to claim 15 , wherein the heterodimeric Fc portion is connected via a disulfide bond in the hinge region and a knob-in-hole structure in the CH3 domain.

17. The fusion protein according to any one of claims 1 to 16, wherein the CD3 binding portion comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3 or IgG4. The fusion protein according to claim 17 , wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

17.

19. The fusion protein of any one of claims 1-18, wherein the CD3 binding portion comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region or a human Igλ constant region.

20. The fusion protein of claim 19, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

20.

21. The fusion protein of any one of claims 1 to 20, wherein the CD3 binding portion comprises an antibody heavy chain and a light chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

21.

22. The fusion protein of any one of claims 1-21, wherein the CD47 binding portion comprises an antibody heavy chain constant region, and the antibody heavy chain constant region comprises a constant region derived from human IgG1, IgG2, IgG3 or IgG4.

23. The isolated fusion protein of claim 22, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

47.

24. The fusion protein of any one of claims 1-23, wherein the CD47 binding portion comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

48.

25. A fusion protein comprising two heavy chains and one light chain, wherein the first heavy chain comprises VH-CH1-hinge region-Fc from N-terminus to C-terminus, and the first light chain comprises VL-CL from N-terminus to C-terminus; the second heavy chain comprises SIRPα-hinge region-Fc from N-terminus to C-terminus, wherein the VH-CH1 of the first heavy chain and the VL-CL of the first light chain form an antigen binding site that binds to CD3, and the SIRPα of the second heavy chain binds to CD47; wherein the first heavy chain comprises three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3), the first light chain comprises three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), wherein HCDR1 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 4, 33, 41 and 49; wherein HCDR2 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 5, 34, 42 and 50; wherein HCDR3 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 6, 35, 43 and 51, wherein LCDR1 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 1, 36, 44 and 52, wherein LCDR2 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 2, 37, 45 and 53, and wherein LCDR3 comprises the amino acid sequence as shown in any one of SEQ ID NOs: 3, 38, 46 and 54; and wherein the second heavy chain comprises SIRPα, wherein the SIRPα comprises the amino acid sequence as shown in SEQ ID NO:

46.

26. The fusion protein of claim 25, wherein the first heavy chain comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and the first light chain comprises light chain variable regions LCDR1, LCDR2, and LCDR3; wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:6; and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:3; or; wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 25; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 26, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 27, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29; or; wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32; wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33; wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 35, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 36, and wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 37; Or; wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 comprises the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 comprises the amino acid sequence shown in SEQ ID NO:42, wherein LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43, wherein LCDR2 comprises the amino acid sequence shown in SEQ ID NO:44, and wherein LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

45.

27. The fusion protein of any one of claims 25-26, wherein the first heavy chain comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NOs: 8, 15, 16, 22, 30 and 38; and / or the first light chain comprises a light chain variable region, and the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID NOs: 7, 19, 23, 31 and 39.

28. The fusion protein according to any one of claims 25 to 27, wherein the first heavy chain comprises a heavy chain variable region, and the first light chain comprises a light chain variable region; wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19; Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23; Or; the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31; or Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

39.

29. The fusion protein of any one of claims 25-28, wherein the first heavy chain and the second heavy chain comprise Fc from IgG. The fusion protein according to claim 29 , wherein the Fc is an Fc from IgG1, IgG2, IgG3 or IgG4.

31. The fusion protein of any one of claims 25-30, wherein the first heavy chain and the second heavy chain comprise Fc from human IgG. The fusion protein according to claim 31 , wherein the Fc is an Fc from human IgG1, human IgG2, human IgG3 or human IgG4.

33. The fusion protein according to any one of claims 25 to 32, wherein the first heavy chain and the second heavy chain are connected by a disulfide bond in the hinge region and a knob-in-hole structure in the CH3 domain.

34. The fusion protein of any one of claims 25-33, wherein the first heavy chain and the second heavy chain are human IgG1 isotypes, and wherein one of the first heavy chain or the second heavy chain comprises T366S, L368A, and Y407V heavy chain substitutions, and the other of the first heavy chain or the second heavy chain comprises a T366W heavy chain substitution, wherein the residues are numbered according to the EU index.

35. The fusion protein of any one of claims 25-34, wherein the first light chain comprises a CL from a human lambda or kappa light chain.

36. The fusion protein according to any one of claims 25 to 35, wherein the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 18, and the first light chain comprises the amino acid sequence shown in SEQ ID NO:

21.

37. The fusion protein of any one of claims 25-36, wherein the second heavy chain comprises the amino acid sequence shown in SEQ ID NO:

48.

38. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-37.

39. A vector comprising the nucleic acid according to claim 38.

40. A host cell comprising the expression vector according to claim 39. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 37 and a pharmaceutically acceptable carrier.

42. Use of the fusion protein of any one of claims 1-37, the isolated nucleic acid molecule of claim 38, the vector of claim 39, the host cell of claim 40, or the pharmaceutical composition of claim 41 in the preparation of a medicament for treating cancer.

43. The use according to claim 42, wherein the cancer comprises solid tumors and hematological tumors.

44. The use according to claim 42, wherein the cancer is a CD47 expressing cancer.

45. The use according to claim 42, wherein the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.

46. ​​A method of treating cancer in a subject, the method comprising administering to the subject the fusion protein of any one of claims 1-37, the nucleic acid molecule of claim 38, the vector of claim 39, the host cell of claim 40, or the pharmaceutical composition of claim 41, thereby inhibiting growth of the cancer in the subject.

47. The method of claim 46, wherein the cancer comprises solid tumors and hematological tumors.

48. The method of claim 46, wherein the cancer is a CD47-expressing cancer.

49. The method of claim 48, wherein the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.

50. The method of any one of claims 46-49, further comprising administering to the subject a second therapeutic agent.

51. The method of claim 50, wherein the second therapeutic agent is an anti-tumor agent, radiation therapy, an antibody drug conjugate, a checkpoint inhibitor, or a combination thereof.

52. A method for producing the fusion protein of any one of claims 1-37, wherein the method comprises culturing the host cell of claim 40 under conditions capable of expressing the fusion protein.

53. The method of claim 52, wherein the host cell is selected from a bacterial cell, a fungal cell, a plant cell, a mammalian cell, or a virus; The method according to claim 52, wherein the bacterial cell is Escherichia coli; and wherein the fungal cell is a yeast cell; The mammalian cells are selected from CHO, NS0, BHK or HEK293 cells.

54. The method of claim 52, wherein the cell is a hybridoma cell.

55. The method of claim 54, wherein the hybridoma cell is selected from mouse, rat, or rabbit.

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