WO2024061350A1

WO2024061350A1 - Fusion protein and use thereof

Info

Publication number: WO2024061350A1
Application number: PCT/CN2023/120732
Authority: WO
Inventors: 孙哲; 杨颖�
Original assignee: 广州凌腾生物医药有限公司
Priority date: 2022-09-23
Filing date: 2023-09-22
Publication date: 2024-03-28

Abstract

Provided is a fusion protein consisting of SIRPαV2 and a CD3 antibody.

Description

Fusion proteins and their applications

The present invention claims the priority of Chinese patent application CN202211163564.8 submitted on September 23, 2022. The description, drawings and claims of the priority document are fully incorporated into the description of the present invention and regarded as the present invention. Part of the original instructions. The applicant further declares that the applicant has the right to amend the description and claims of the invention based on the priority document.

Technical field

This application relates to the field of biomedicine, specifically to a fusion protein composed of SIRPαV2 and CD3 antibody.

Background technique

Fusion proteins can simultaneously recognize two different targets and influence the activation of more cell activation regulatory mechanisms, and have great potential in the treatment of tumors and other diseases. T cell engager bisAb is a typical drug of this type. Its mechanism of action is to form an immune synapse (as shown in Figure 1) by simultaneously binding to T cell surface antigens (such as CD3) and tumor cell surface antigens (such as CD19), thus bringing them closer together. The distance between tumor cells and T cells is directly activated, proliferating T cells, and then using T cells to directly kill tumor cells, or releasing cytotoxins to kill tumor cells. Generally speaking, this 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 transformation rate.

CD47 is a transmembrane protein widely present on the surface of normal cells and belongs to the immunoglobulin superfamily. It is an important target for a new generation of immuno-oncology therapies. CD47 and PD-1 respectively target two major immune cell groups that play a major role in tumor immunotherapy. The CD47 signaling pathway mainly regulates macrophages, and the PD-1 signaling pathway mainly regulates T lymphocytes. Macrophages are an important part of innate immunity and can act as scavengers in the human body by engulfing aging and dead cells. CD47 is highly expressed on a variety of solid tumor cells and malignant hematoma cells, and its expression level is positively correlated with disease progression. Because it can inhibit the phagocytosis of tumor cells by macrophages after binding to SIRPα, it is usually overexpressed on the surface of tumor cells to assist tumor cells in immune escape. CD47 pathway drugs can theoretically be used in the treatment of many types of cancers like PD-1 drugs. Currently, CD47-targeting drugs have been used as single agents and combination treatments in the treatment of common blood tumors and solid tumors such as leukemia, lymphoma, lung cancer, and liver cancer. Under treatment.

Contents of the invention

There are three known natural ligands of CD47: integrins, platelet agglutinin-1 and SIRPα. The biological effects it participates in include: cell adhesion, cell migration, regulation of inflammatory responses and inhibition of macrophage phagocytosis. In view of the above-mentioned immune checkpoint functions of CD47 and SIRPα, the use of SIRPαV domain in molecular design can help target CD47 and relieve the resistance of cancer cells by preventing CD47 on the surface of cancer cells from binding to SIRPα on the surface of macrophages or DC cells. The inhibitory effect of macrophages induces the phagocytosis of tumor cells by macrophages, promotes the uptake of tumor cells by DC cells, and is beneficial to the presentation of tumor antigens.

Since CD47 is also expressed on the surface of normal cells (especially red blood cells) and the SIRPαV2 SNP variant shows a selective weak affinity for CD47 on the surface of red blood cells, in order to avoid off-target toxicity, the selection of SIRPαV2 variants in molecular design can greatly alleviate cell agglutination and lysis, and thrombocytopenia and other issues.

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

On the one hand, the application provides an isolated fusion protein including a CD3 binding portion and a CD47 binding portion;

wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is any one of SEQ ID NO: 4, 24, 32 and 40 The amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO: 6, 26, 34 and 42 Item 1, and/or the CD3 binding portion includes 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 such as SEQ ID NO: 1, 27, As shown in any one of 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NO:2, 28, 36 and 44; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3, 29, 37 and 45, wherein the CD3 binding moiety is capable of inducing T cell activation.

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.

In certain embodiments, the CD47 ligand comprises integrin, thromboagglutinin-1, SIRPγ, SIRPα or functional variants or fragments thereof.

In certain embodiments, wherein the CD47 binding moiety includes SIRPα, or a functional variant thereof, or a fragment thereof.

On the other hand, the present application provides an isolated fusion protein including a CD3 binding portion and a CD47 binding portion;

wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is any one of SEQ ID NO: 4, 24, 32 and 40 The amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO: 6, 26, 34 and 42 Item 1, and/or the CD3 binding portion includes 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 such as SEQ ID NO: 1, 27, As shown in any one of 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NO:2, 28, 36 and 44; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3, 29, As shown in any one of 37 and 45, wherein the CD3 binding portion can induce T cell activation;

Wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with the SIRPα protein, the amino acid sequence of the SIRPα protein is shown in SEQ ID NO: 46.

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 the reference antigen binding protein for human CD3 Binding, the reference antigen binding protein includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), wherein HCDR1 contains as shown in SEQ ID NO:4 The amino acid sequence of HCDR2 includes the amino acid sequence shown in SEQ ID NO:5; HCDR3 includes the amino acid sequence shown in SEQ ID NO:6, and LCDR1 includes the amino acid sequence shown in SEQ ID NO:1, where LCDR2 includes the amino acid sequence set forth in SEQ ID NO:2, and wherein LCDR3 includes the amino acid sequence set forth in SEQ ID NO:3; and

The CD47 binding part competes with the SIRPα protein for binding to human CD47, and the SIRPα protein includes the amino acid sequence shown in SEQ ID NO: 46.

On the other hand, the present application provides an isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion, wherein the CD3 binding portion includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementation determining regions. Region (LCDR1, LCDR2 and LCDR3), wherein HCDR1 includes the amino acid sequence shown in any one of SEQ ID NO:4, 24, 32 and 40; wherein HCDR2 includes any one of SEQ ID NO:5, 25, 33 and 41 The amino acid sequence shown in one item; wherein HCDR3 includes the amino acid sequence shown in any one of SEQ ID NO:6, 26, 34 and 42, and wherein LCDR1 includes any one of SEQ ID NO:1, 27, 35 and 43. The amino acid sequence shown in one item, wherein LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO:2, 28, 36 and 44, and wherein LCDR3 includes any one of SEQ ID NO:3, 29, 37 and 45. an amino acid sequence represented by an item; and

The CD47 binding part includes the amino acid sequence shown in SEQ ID NO: 46.

In certain embodiments, 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 set forth in SEQ ID NO: 4 As shown, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:5 and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:6; and/or the amino acid sequence of the LCDR1 is as shown in SEQ ID NO:1, The amino acid sequence of LCDR2 is shown in SEQ ID NO:2 and the amino acid sequence of LCDR3 is shown in SEQ ID NO:3;

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

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

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

In certain embodiments, wherein the CD3 binding moiety comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is any one of SEQ ID NOs: 8, 15, 16, 22, 30 and 38 As shown in the item; and/or the CD3 binding portion includes 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 NO: 7, 19, 23, 31 and 39 .

In certain embodiments, 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 set forth in SEQ ID NO: 8, and the light chain The variable region contains the amino acid sequence shown in SEQ ID NO:7;

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;

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

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

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

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.

In certain embodiments, the CD3-binding portion is an antibody or antigen-binding fragment thereof.

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

In certain embodiments, wherein the antigen-binding fragments include Fab, Fab', Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb.

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

In certain embodiments, wherein the Fc is from an Fc of IgGl, IgG2, IgG3 or IgG4.

In certain embodiments, the heterodimeric Fc portion is connected by a disulfide bond in the hinge region and a hinge structure of the CH3 domain.

In certain embodiments, 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 IgGl, IgG2, IgG3 or IgG4.

In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 17.

In certain embodiments, 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.

In certain embodiments, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20.

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.

In certain embodiments, 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 IgGl, IgG2, IgG3 or IgG4.

In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 47.

In certain embodiments, wherein the CD47 binding portion comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 48.

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 VH-CH1-hinge region-Fc from N-terminus to C-terminus. It has VL-CL from end to C end; the second heavy chain has SIRPα-hinge region-Fc from N end to C end, wherein the VH-CH1 of the first heavy chain and the VL-CL of the first light chain form The antigen binding site binds to CD3, and the SIRPα of the second heavy chain binds to CD47;

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

In certain embodiments, 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 SEQ ID NO:4 is shown, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:5 and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:6; and/or the amino acid sequence of the LCDR1 is shown in SEQ ID The amino acid sequence of the LCDR2 is shown in SEQ ID NO:2 and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:3;

Wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO:24; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:25; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:26, wherein LCDR1 includes the amino acid sequence shown in SEQ ID NO:25 The amino acid sequence shown in ID NO:27, wherein LCDR2 includes the amino acid sequence shown in SEQ ID NO:28, and wherein LCDR3 includes the amino acid sequence shown in SEQ ID NO:29;

wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO:32; wherein HCDR2 contains the amino acid sequence shown in SEQ ID NO:33; wherein HCDR3 contains the amino acid sequence shown in SEQ ID NO:34, and LCDR1 contains the amino acid sequence shown in SEQ The amino acid sequence shown in ID NO:35, wherein LCDR2 includes the amino acid sequence shown in SEQ ID NO:36, and wherein LCDR3 includes the amino acid sequence shown in SEQ ID NO:37; or

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

In certain embodiments, wherein the first heavy chain comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is any of SEQ ID NOs: 8, 15, 16, 22, 30 and 38. as shown in one item; 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 in any one of SEQ ID NO: 7, 19, 23, 31 and 39 shown.

In certain embodiments, wherein the first heavy chain includes a heavy chain variable region, the first light chain includes a light chain variable region; wherein the heavy chain variable region includes SEQ ID NO: 8 The amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO:7;

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

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

In certain embodiments, wherein the first heavy chain and the second heavy chain comprise Fc from IgG.

In certain embodiments, wherein the Fc is Fc from IgGl, IgG2, IgG3 or IgG4.

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

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

In certain embodiments, the first heavy chain and the second heavy chain are connected through a disulfide bond in the hinge region and a pestle structure of the CH3 domain.

In certain embodiments, 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 contains the T366W heavy chain substitution, where the residues are numbered according to the EU index.

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

In some 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.

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

In another aspect, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein described in the present application or a fusion protein described in the present application.

The nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplification in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) production by clonal recombination, (iii) purification , for example by enzymatic digestion and gel electrophoresis fractionation, or (iv) synthetic, for example by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

In this 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, using restriction fragment manipulation or using synthetic oligonucleotides. For overlap extension PCR, specific procedures can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

In another aspect, the present application provides a vector comprising a nucleic acid according to the present application. One or more such nucleic acid molecules may be included in each vector. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may contain expression control elements that allow correct 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 sequences are tunable elements. The specific structure of the expression control sequence can 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 the initiation of transcription and translation, respectively, such as TATA boxes, GA cap sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may comprise a promoter region, which may comprise a promoter sequence for transcriptional control of a functionally linked nucleic acid. The expression control sequences may also include enhancer sequences or upstream activator sequences. In this application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters thereof (such as CMV), where the promoter A certain part can be fused with a certain part of the gene promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not include additional introns. One or more nucleic acid molecules described herein can be operably linked to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

In another aspect, the present application provides a cell comprising a vector according to the present application. In certain embodiments, each or each host cell contains one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell contains multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. The vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

On the other hand, the present application provides a pharmaceutical composition, which includes the fusion protein described in the present application and a pharmaceutically acceptable carrier.

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 drug formulation 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, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other auxiliary agents. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The active ingredients are administered in amounts that are therapeutically effective. In addition, the fusion proteins described herein can also be used with other therapeutic agents.

The fusion proteins or pharmaceutical compositions described herein may be formulated, administered, and administered in a manner consistent with good medical practice. Considerations in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the etiology of the condition, the site of agent delivery, the method of administration, and other factors known to the medical practitioner. The therapeutic agent need not be, but optionally, be formulated and/or administered concurrently with one or more agents currently used to prevent or treat the condition under consideration. The effective amount of such other agents depends on the amount of therapeutic agent present in the formulation, the type of condition or treatment, and other factors discussed above. These agents can generally be used at any dose and by any route empirically/clinically determined to be appropriate. The dose of antibodies administered in combination treatment can be reduced compared to individual treatments. The progress of this therapy is easily monitored by conventional techniques.

On the other hand, the present application provides the fusion protein described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the host cell described in the present application, or the pharmaceutical composition described in the present application. Use in the preparation of medicaments for the treatment of cancer.

In certain embodiments, the cancer includes solid tumors and hematological tumors.

In certain embodiments, wherein the cancer is a CD47-expressing cancer.

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

On the other hand, the present application provides a method for treating cancer in a subject, the method comprising administering to the subject the fusion protein described in the present application, the isolated nucleic acid molecule described in the present application, the 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.

In certain embodiments, wherein the cancer is a CD47-expressing cancer.

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

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

In certain embodiments, wherein the second therapeutic agent is an antineoplastic agent, radiation therapy, an antibody drug conjugate, a checkpoint inhibitor, or a combination thereof.

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.

In certain embodiments, the host cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, mammalian cells, or viruses.

In certain embodiments, wherein the bacterial cell is E. coli.

In certain embodiments, wherein the fungal cell is a yeast cell.

In certain embodiments, wherein the mammalian cells are selected from CHO, NSO, BHK or HEK293 cells.

In certain embodiments, the cells are hybridoma cells.

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

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description below. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will realize, the contents of this application enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention covered by this application. Accordingly, the drawings and descriptions of the present application are illustrative only and not restrictive.

Description of drawings

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

Figure 1 shows the configuration diagrams of the asymmetric anti-CD3×SIRPa-Fc Fusion Protein (D1-TW) and symmetric anti-CD3 x SIRPa (H3S02) described in this application.

Figure 2 shows the vector p2MPT map.

Figure 3 shows the vector pMPTN map.

Figure 4 shows a two-step purification diagram of the Mos-D1-TW molecule.

Figure 5 shows the SDS-PAGE image after purification of Mos-D1-TW molecules.

Figure 6 shows the two-step purification diagram of the D1-TW molecule.

Figure 7 shows the SDS-PAGE image after purification of D1-TW molecules.

Figure 8 shows the purification diagram of H3S02 molecule.

Figure 9 shows the SDS-PAGE image after purification of H3S02 molecules.

Figure 10 shows the affinity check results of D1-TW with CD3E/G and CD47.

Figure 11 shows the ELISA detection results of Mos-D1-TW double antibody.

Figure 12 shows the FACS detection of the binding of D1-TW to 8226 and HCT-8 tumor cells.

Figure 13 shows the FACS detection of the binding of mos-D1-TW to target cells 8226 and hCD3E transgenic mouse PBMC.

Figure 14 shows the killing effect of hPBMC on D1-TW-mediated tumor cells 8226 and HCT-8.

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

Detailed ways

The implementation of the invention of the present application will be described below with specific examples. Those familiar with this technology can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

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.

In order to make the present invention more easily understood, certain scientific and technical terms are specifically defined as follows. Unless otherwise expressly defined in other parts of this document, the scientific and technical terms used in this document have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs. Regarding the definitions and terms in this field, 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 expressly provided in the text.

In this application, the term "about" when used in conjunction with a numerical value is intended to encompass a range of numerical values 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.

In this application, the term "and/or" shall be understood to mean any one of the optional items or any combination of two or more of the optional items.

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, 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. It is then connected to the intracellular inhibitory signaling domain through a transmembrane helical domain. In this application, the term "CD3" generally refers to being part of a T cell receptor complex consisting of three distinct chains, CD3ε, CD3δ and CD3γ. The concentration of CD3 on T cells, produced by, for example, its immobilization by anti-CD3 antibodies, results in activation of T cells that is similar to T cell receptor-mediated activation but is independent of the specificity of the TCR clone. The vast majority of anti-CD3 antibodies recognize the CD3 epsilon chain. The term refers to any native CD3 from any vertebrate (including mammals such as primates (eg, humans)) and rodents (eg, mice and rats), unless otherwise stated. The term encompasses "full-length" unprocessed CD3 as well as any form of CD3 resulting from intracellular processing or any fragment thereof. The term also includes naturally occurring variants of CD3, eg, splice variants or allelic variants. In a preferred embodiment, CD3 refers to the full length CD3 from human and cynomolgus monkey or a fragment thereof (such as its mature fragment lacking a signal peptide). In a preferred embodiment, CD3 refers to the full length from mouse/rat or a fragment thereof (such as its mature fragment lacking a signal peptide).

In this application, the term "percent (%) amino acid sequence identity" or simply "identity" is defined as when the amino acid sequences are aligned (and gaps introduced when necessary) to obtain the maximum percent sequence identity without The percentage of amino acid residues in the candidate amino acid sequence that are identical to the amino acid residues in the reference amino acid sequence after any conservative substitutions are considered part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed 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 algorithms required to obtain maximal alignment over the full length of the sequences being compared.

In this application, the term "functional variant" generally refers to a nucleotide comprising an alteration of 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. and/or amino acid sequence of a nucleic acid molecule or protein. 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, that is, the binding molecule is still able to recognize and bind to it. target. Functional variants of a gene include gene variants with minor changes, such as silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions that do not significantly alter the function of the gene. Functional variants can have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can include natural as well as unnatural nucleotides and amino acids.

In this application, the term "immune response" generally refers to the effect of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and the production of soluble macromolecules (including antibodies, cytokines and complements) by these cells or the liver, which The effects result in the selective damage, destruction or removal from the human body of invading pathogens, pathogen-infected cells or tissues, cancer cells or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

As used herein, the term "signal transduction pathway" or "signal transduction activity" refers to a biochemical causal relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in a signal emanating from a part of the cell. passed to another part of the cell. Generally, transmission involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a cascade of reactions that results in signal transduction. Penultimate processes often involve nuclear events leading to changes in gene expression.

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 targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics are observed, measured or evaluated using techniques known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays without limitation, receptor binding, production and/or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates or any other source).

As used herein, the term "fusion protein" generally refers to an amino acid sequence containing a first polypeptide or protein, or a fragment, analog or derivative thereof, and a heterologous polypeptide or protein (i.e., different from the first polypeptide or protein or of a second polypeptide or protein or a fragment, analog or derivative thereof, or that is generally not part of the first polypeptide or protein or a fragment, analog or derivative thereof) of the amino acid sequence Peptide or protein. In some cases, a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide. Wherein, the heterologous protein, polypeptide or peptide may or may not be a preventive or therapeutic drug of different types. For example, two different proteins, polypeptides or peptides with immunomodulatory activity can be fused together to form a fusion protein. In some cases, the fusion protein retains or has improved activity compared to the activity of the heterologous protein, polypeptide, or original polypeptide or protein prior to fusion.

As used herein, the term "antigen-binding protein" generally refers to a protein comprising an antigen-binding portion, and optionally a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes 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 fragment, F(ab')2, scFv, di-scFv and/or dAb), immunoconjugates Objects, multispecific antibodies (such as fusion proteins), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they display the required antigen-binding activity. An "isolated antigen-binding protein" of the present application may comprise an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding of the antigen-binding portion by the antigen-binding portion.

In this application, the term "antibody" generally refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is 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 consists of tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). 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 the constant region primarily responsible for effector function. Human light chains are generally classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, where the heavy chain also includes a "D" region of about 10 or more amino acids. See generally Chapter 7 of Fundamental Immunology (Ed. Paul, W., 2nd ed. Raven Press, N.Y. (1989)).

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

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target 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 characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

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

As used herein, the term "full-length antibody" generally refers to an immunoglobulin molecule that when naturally occurring contains four peptide chains: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains. ) chains (approximately 25 kDa in full length) are connected to each other by disulfide bonds. Each heavy chain consists 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 consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conservative regions called framework regions (FRs). Each VH or VL region consists of 3 CDRs and 4 FRs arranged from the amino terminus to the 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 the antigen. The constant region of an antibody may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

In this application, the term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of the antibody, typically including at least one of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody Fragments that retain 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 formed from antibody fragments and multispecific antibodies. When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally 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 greater of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functionally conserved variants" of the antibody). The term "binding compound" refers to both an antibody and its binding fragment.

In this application, 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 also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

In this application, the term "complementarity determining region (CDR)" is the region of an antibody that binds an antigen. CDR can be defined using various depictions, 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, JBmol Biol, Vol. 263, pp. 800-815). Correspondences between various depictions and variable region numbers are described (see e.g. Lefranc et al., 2003, Dev CompImmunol, vol. 27, pp. 55-77; Honegger and Pluckthun, 2001, J Mol Biol, vol. Volume 309, pages 657-670; International Immunogenetics (IMGT) database; Web resource, http://www_imgt_org). Available programs such as UCL Business PLC's abYsis can be used to characterize CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include data produced by any of the above methods (Kabat, Chothia, IMGT or AbM) defined CDR. For example, the CDR region of this application can be defined using Kabat rules.

In this application, the term "Fc" or "Fc region" or "Fc fragment" generally refers to the CH2 and CH3 domains of IgA, IgD and IgG, or the CH2, CH3 and CH4 domains of IgE and IgM through the hinge. region of the polypeptide. Although the fragmentation of the Fc fragment is variable, the heavy chain Fc fragment of human IgG generally refers to the stretch of polypeptide from A231 to its carboxyl terminus.

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 recognized hinge region of IgG is a polypeptide chain composed of amino acid residues from 216 to 230.

In this application, the term "domain antibody" generally refers to an immunologically functional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked to 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.

In this application, the term "bivalent antibody" encompasses 2 antigen-binding sites. In some cases, 2 binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.

In this application, the term "diabody" generally refers to a small antibody fragment with two antigen-binding sites contained in the same polypeptide chain (VH-VL or VL-VH) with a light chain variable domain (VH-VL or VL-VH). VL) linked heavy chain variable domain (VH). By using a linker that is too short to allow pairing between two domains of the same chain, this domain is forced to pair with a complementary domain of the other chain and creates two antigen-binding sites.

In this application, the term "chimeric antibody" generally refers to an antibody having a variable domain of a first antibody and a constant domain of a second antibody, where the first and second antibodies are from different species. Typically, the variable domains are obtained from an antibody such as a rodent (a "parent antibody"), and the constant domain sequences are obtained from a human antibody such that the resulting chimeric antibody induces in human subjects compared to the parent rodent antibody. There is a lower likelihood of adverse immune responses.

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 a minimal non-human (e.g., mouse) sequence. Typically, a humanized antibody is a human immunoglobulin, wherein the residues from the complementary determining region (CDR) are replaced by residues from the CDR of a 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.

In this application, the term "fully human antibody" generally refers to an antibody that contains only human immunoglobulin protein sequences. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, fully human antibodies may contain mouse sugar chains. Similarly, "mouse antibodies" refer to antibodies that contain only mouse immunoglobulin sequences. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, fully human antibodies may contain rat sugar chains. Similarly, "rat antibodies" refer to antibodies that contain only rat immunoglobulin sequences.

In this application, "isotype" of an antibody generally refers to the class of antibody provided by the heavy chain constant region gene (eg, IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been made to alter Fc function, for example to enhance or weaken effector function or binding to Fc receptors.

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

In this application, "affinity" or "binding affinity" generally refers to the inherent 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 used to measure affinity is the ForteBio kinetic binding assay described herein.

In this application, the term "does not bind" to a protein or cell generally means that it does not bind to the protein or cell, or does not bind to it with high affinity, that is, the KD of the binding protein or cell is 1.0×10 ^-6 M or higher, More preferably, it is 1.0×10 ^-5 M or higher, more preferably 1.0×10 ^-4 M or higher, 1.0×10 ^-3 M or higher, and more preferably 1.0×10 ^-2 M or higher.

In this application, the term "high affinity" for IgG antibodies generally means that the KD for the antigen is 1.0×10 ^-6 M or lower, preferably 5.0×10 ^-8 M or lower, and more preferably 1.0 ×10 ^-8 M or less, 5.0 × 10 ^-9 M or less, more preferably 1.0 × 10 ^-9 M or less. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of the IgM subtype means a KD of 10 ^"6 M or less, preferably 10" ⁷ M or less, more preferably 10" ⁸ M or less.

As used herein, the terms "antibody-dependent cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refer to cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens to Target cells to which the antibody binds, such as cancer cells, are lysed.

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

In this application, the term "nucleic acid" or "polynucleotide" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form. Unless expressly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, belonging to Kariko et al. Human U.S. Patent No. 8,278,036, which discloses an mRNA molecule in which uridine is replaced by pseudouridine, methods of synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences thereof as well as the sequences explicitly indicated. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or a deoxyinosine residue (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)).

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

As used herein, "vector" generally refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., the introduction of heterologous DNA into a host cell). One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell and thus replicate with the host genome. In addition, certain vectors are capable of directing expression of operably linked genes. Such vectors are referred to herein as "expression vectors."

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

In this application, "activation," "stimulation," and "treatment" of a cell or receptor may have the same meaning, for example, a cell or receptor is activated, stimulated, or treated with a ligand, unless the context otherwise or expressly dictates otherwise. "Ligands" include natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules such as peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cellular activation regulated by internal mechanisms as well as external or environmental factors. A "response", such as a response of a cell, tissue, organ or organism, includes a change in biochemical or physiological behavior (such as concentration, density, adhesion or migration, gene expression rate or differentiation status within a biological compartment) in which the change Relating to activation, stimulation or processing, or to internal mechanisms such as genetic programming.

In this application, the term "treating" or "treating" any disease or condition in one embodiment means ameliorating the disease or condition (ie, slowing or arresting or reducing at least one of the progression of the disease or its clinical symptoms). In another embodiment, "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discernible by the patient. In another embodiment, "treating" or "treating" means modulating a disease or disorder physically (eg, stabilization of discernible symptoms), physiologically (eg, stabilization of body parameters), or both. Unless explicitly described herein, methods for assessing treatment and/or prevention of disease are generally known in the art.

As used herein, "subject" includes any human or non-human animal. The term "non-human animals" includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like. As used herein, the term "cyno" or "cyno" refers to the cynomolgus monkey.

As used herein, administration "in conjunction with" one or more other therapeutic agents includes simultaneous (co) administration and sequential administration in any order.

In this application, "therapeutically effective amount", "therapeutically effective dose" and "effective amount" generally refer to the ability of the antigen-binding protein of the present invention to effectively prevent or an amount that ameliorates the symptoms of one or more diseases or conditions or the progression of that 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 that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition. When an active ingredient is administered to an individual when administered alone, the therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether combined, administered sequentially, or administered simultaneously. An effective amount of the therapeutic agent will result in an improvement in diagnostic criteria or parameters of at least 10%; typically at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.

As used herein, "cancer" and "cancerous" generally refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. 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 gastric 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 or kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin's disease) King's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small anucleate cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-associated lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as those associated with phakomatoses, edema (such as those associated with brain tumors), and leucocytosis Abnormal vascular proliferation associated with Meigs syndrome.

Without intending to be limited by any theory, the following examples are only to illustrate the fusion protein of the present application, preparation methods and uses, etc., and are not intended to limit the scope of the invention of the present application.

Example

1. Antibody expression

The sequences encoding the asymmetric structure of the Linton anti-CD3 antibody heavy chain and the Linton anti-CD3 antibody light chain were inserted into the EcoRI/XbaI expression cassette and NotI/BamHI expression cassette of the vector p2MPT, respectively, to form the recombinant plasmid 1p2MPT-hDL-D1TW-GA; The sequence encoding SIRPa-Fc Fusion protein was connected to the NotI/BamHI expression cassette of the vector p2MPT to form recombinant plasmid 2 p2MPT-hS1-GA. After gene sequencing and enzyme digestion were verified, the QIAGEN Plasmid Midi Kit was used to prepare the correctly constructed plasmid 1 and plasmid 2.

The sequences encoding the competitor anti-CD3 antibody heavy chain and the competitor anti-CD3 antibody light chain are each connected to the NotI/BamHI expression cassette of the vector pMPTN to form recombinant plasmid 3pMPTN-MD1TW-HC and plasmid 4pMPTN-MD1TW-LC respectively. After verification by gene sequencing and enzyme digestion, the Endo-Free PlasmidMaxi Kit (Omega) kit was used to prepare the correctly constructed plasmid 3 and plasmid 4.

The sequences encoding the heavy chain and light chain of the anti-CD3 x SIRPa antibody with a symmetrical structure were connected to the NotI/BamHI expression cassette and EcoRI/XbaI expression cassette of the vector p2MPT respectively, forming recombinant plasmid 5 p2MPT-H3S02-HC-C3002LC. After verification by gene sequencing and enzyme digestion, the QIAGEN PlasmidMidi Kit was used to prepare the correctly constructed plasmid 5.

When asymmetric Linton anti-CD3 x SIRPa-Fc Fusion Protein (D1-TW) and symmetric Linton anti-CD3 x SIRPa (H3S02) are expressed, CHO-DG44-express stored in liquid nitrogen is recovered and cultured (Ex- cell Advanced CHO Fed-Batch Medium, Sigma), culture conditions were 37°C, 180 rpm, 75% RH, 5% CO2. The transfection and expression operation was performed after the cell viability stabilized to above 98%. During transfection, the cell density was 3.0×10 ⁶ cells/ml, the transfection medium was proCHO5 medium (Lonza), the transfection reagent was 25K polyethylenimine (PEI, sigma), and the DNA dosage was 3ug/mL (cell Culture volume, the dosage of helper plasmid pBase is 10% of the total DNA amount), and the dosage ratio to PEI is 1:3. The dosage of Linton anti-CD3 plasmid and SIRPa-Fc Fusion plasmid is 1:1. 24 hours after transfection, the medium was replaced with Ex-cell Advanced CHO Fed-Batch Medium (Sigma), and puromycin (Enzo) was used for screening. During screening, the seeding cell density was 5*10^5 cells/mL, and the final concentration of puromycin was is 10ug/mL. When the cell viability stably recovers to above 98%, antibody expression is performed. After expression for 7 days at 31°C, 180 rpm, 75% RH, 5% CO2, the supernatant was collected by centrifugation.

When expressing asymmetric Competitor anti-CD3 x SIRPa-Fc Fusion Protein, Expi293F (gibco) cells stored in liquid nitrogen were recovered and cultured (Wayne 293 Transfection Medium, Kang Tianshenghe). The culture conditions were 37°C, 110 rpm, 75% RH, and 6% CO2. The transfection and expression operation was performed after the cell viability stabilized to above 98%. During transfection, the cell density is 1.5×10 ⁶ cells/ml, the transfection reagent is 25K polyethylenimine (PEI, sigma), the DNA dosage is 1ug/mL (cell culture volume), and the dosage ratio to PEI is 1: 4. The dosage ratio of Competitor anti-CD3 heavy and light chain plasmid and SIRPa-Fc Fusion plasmid is 1:1:1. On the 6th day after transfection, the supernatant was collected by centrifugation.

2. Antibody enrichment

Take the CHO cell expression supernatant, centrifuge at 2000 rpm to remove cells, and centrifuge at 8000 rpm to remove cell debris. Filter with a 0.45 μm microporous filter to obtain a clarified culture medium. After equilibrating the Mabselect Sure column with the PBS solution, fully enrich the Fc-end antibody in the CHO cell expression supernatant according to the retention time of 4 min/min. After the PBS solution equilibrates the column again, wash it with 20mM sodium citrate buffer (pH3.0). Remove, collect the samples, and adjust the samples to pH 5.6 with Tris-HCl (pH 8.5).

3. Double Antibody Pure

3.1 Ion chromatography

Mos-D1TW molecules were purified using cation chromatography. Equilibrate the SPHP column with 20mM sodium citrate buffer (pH 5.4), and then flow the enriched antibody solution through the SPHP column at 1 ml/min. After the injection is completed, continue to use 20mM sodium citrate buffer (pH 5.4) to equilibrate the column. Finally, linear elution from A: 20mM sodium citrate buffer (pH5.4) to 35% B: 20mM sodium citrate buffer, 1M NaCl (pH5.4), 20CV, samples were collected respectively, and peak 1 (approximately 110kda), peak 2 (impurity).

3.2 Hydrophobic chromatography

The D1-TW molecule was purified using a hydrophobic column. The UniHR Phenyl-30L column was equilibrated with 50mM sodium phosphate, 1M ammonium sulfate (pH6.8) buffer, and then the enriched antibody solution was added with 2M ammonium sulfate (pH6.8) at a ratio of 1:1, mixed and flowed through the UniHR Phenyl-30L column at 1ml/min. After the injection, the column was equilibrated with 50mM sodium phosphate buffer, 1M ammonium sulfate (pH6.8). Finally, linear elution was performed from A: 50mM sodium phosphate, 1M ammonium sulfate (pH6.8) to 100% B: 50mM sodium phosphate buffer, 20CV, and samples were collected separately to obtain peak 1 (about 120kda) and peak 2 (regeneration).

4. In vitro functional activity analysis

4.1 Elisa assay for D1-TW activity

Use 0.05M CB solution to coat CD47-hFc (Yiqiao, 12283-H02H) 1.5ug/ml, overnight at 4°C; wash 3 times with PBST the next day, add 1xPBS+1% BSA Elisa buffer to block, 200ul per well. Place it in a 37°C incubator for 1 hour; prepare the sample D1-TW to be tested, dilute D1-TW with Elisabuffer, starting from 9ug/ml, with a 3-fold gradient dilution, including 0 point, a total of 10 concentration points; put the sealed Elisa detection plate Take it out from the incubator, pat it dry, add the diluted antibody, 100ul per well, and place it in a 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, add human CD3E&G-hFc-his (nearshore, C08G), and use Elisabuffer was diluted to 2ug/ml, 100ul per well, and placed in a 37°C incubator for 1 hour; take out the test plate, wash it 3 times with PBST, and add anti-his-HRP (Genscript, A00612, 1:1K), 100ul per well. Place in the 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, add TMB chromogenic solution, 100ul per well, develop color for 10 minutes; add 1M H ₂ SO ₄ , 50ul per well, stop, and read on the microplate reader OD450.

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

4.2 Elisa verifies Mos-D1-TW dual antibody

Coat hCD3E-his (nearshore, C578) 1ug/ml with 0.05M CB solution and keep overnight at 4℃; wash 3 times with PBST the next day, add 1xPBS+1% BSA Elisa buffer to block, 200ul per well, put at 37℃ Incubate for 1 hour; prepare the sample to be tested, Mos-D1-TW, and dilute D1-TW with Elisa buffer. The concentration starts from 10ug/ml and is diluted 10 times, including a total of 5 concentration points at 0; put the sealed Elisa test plate Take it out from the incubator, pat it dry, add the diluted antibody, 100ul per well, and place it in a 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, add anti-SIRPa-mFc (abcam, ab267409), and use Elisa buffer Dilute to 1ug/ml, 100ul per well, and place in a 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, and add Goat anti-mouse Fc-HRP (Jacksonimmuno, 115-035-071, 1:5K). Well 100ul, place in 37°C incubator for 1 hour; take out the detection plate, wash it 3 times with PBST, add TMB chromogenic solution, 100ul per well, develop color for 10 minutes; add 1M H ₂ SO ₄ , 50ul per well, stop, add in enzyme label The meter reads OD450.

As shown in Figure 11, the expressed and purified mos-D-TW is a complete and effective double antibody.

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

Dilute D1-TW with 3% BSA FACS buffer, the starting concentration is 50ug/ml, 3X gradient dilution, including 0ug/ml, a total of 9 concentration points; digest tumor cells HCT-8 with trypsin, and collect 8226 cells at the same time, 200g Centrifuge for 5 minutes, resuspend with 3% BSA, count, adjust the cell density to 2E6 cells/ml, add 100ul per well to a 96-well U-shaped plate, centrifuge at 200g for 5 minutes, remove the supernatant; add the diluted antibody, 100ul per well. , resuspend, and set up a negative control well without adding antibodies, react at 4 degrees for 30 minutes, centrifuge at 200g for 5 minutes, remove the supernatant, and wash with 3% BSA; add fluorescent secondary antibody FITC mouse Anti human IgG 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 in 100ul/well 1xPBS for flow cytometric detection.

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

4.4 FACS detection of mos-D1-TW binding to target cell 8226 and hCD3E transgenic mouse PBMC

Dilute D1-TW with 3% BSA FACS buffer, the starting concentration is 50ug/ml, 5X gradient dilution, including 0ug/ml, a total of 6 concentration points; collect 8226 cells, centrifuge at 200g for 5 minutes, resuspend with 3% BSA, and count , adjust the cell density to 2E6 cells/ml, add 100ul per well to a 96-well U-shaped plate, centrifuge at 200g for 5 minutes, and remove the supernatant; at the same time, take the whole blood of hCD3E transgenic mice, add red blood cell lysis solution to lyse red blood twice, each time 10 minutes, wash twice with 1xPBS, resuspend the cells, add 100ul to each well, centrifuge at 200g for 5 minutes, remove the supernatant; add diluted antibodies, 100ul per well, resuspend, and set up negative control wells without antibodies, 4 degrees React for 30 minutes, centrifuge at 200g for 5 minutes, remove the supernatant, wash once with 3% BSA; add fluorescent secondary antibody FITC mouse Anti human IgG Fc, react at 4 degrees for 30 minutes, centrifuge at 200g for 5 minutes, remove the supernatant, wash once with 1xPBS, and centrifuge at 200g for 5 minutes , remove the supernatant; resuspend in 100ul/well 1xPBS for flow cytometric detection.

The results are shown in Figure 13. Mos-D1-TW has a certain affinity with target cells 8226 and CD3E on the cell surface.

4.5 hPBMCs’ killing effect on D1-TW-mediated tumor cells 8226 and HCT-8

(1)PBMC preparation:

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

(2) Tumor cell 8226 and HCT-8DE preparation:

Take the T75 cell culture flask of normal cultured tumor cells, digest HCT-8 with 2 ml trypsin TRYPSIN 0.25% EDTA, resuspend it with 8 ml cell culture medium, centrifuge at 1000 rpm for 5 minutes, remove the supernatant and wash the cells once with 5 ml culture medium. Remove the supernatant, resuspend with 2 ml culture medium, count cells, adjust the cell density to 1×10^5 cells/ml, add 100ul/well into a 96-well plate containing PBMC, that is, 1×10^4 cells/well, and pre-paste Culture the wall for 2 hours; at the same time, use a pipette to take 10ml of 8226 cells, stain with FAR-red-APC, count the cells, adjust the cell density to 1×10^5 cells/ml, and add 100ul/well to a 96-well plate containing PBMC;

(3) Antibody preparation:

Use culture medium to prepare D1-TW. The starting concentration is 40000ng/ml, 5-fold gradient dilution. Take 50 μl of each concentration of antibody solution into a 96-well plate; then the final concentration is 10000ng/ml, 5-fold gradient dilution, including 0ng/ml, 11 concentration points in total; single wells of PBMC wells and tumor cell wells are set at the same time;

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

(5) For CCK8: After culturing for 24h, 48h, 72h, and 96h respectively in a carbon dioxide incubator (37°C, 5% CO2), the culture supernatant was aspirated for cytokine kit detection; the cultured cells were washed with PBS. Wash away the PBMC several times, then add 100 μl/well of CCK8 working solution, incubate at 37°C for 2 to 4 hours and then measure the OD450; for the 8226 culture system, add 1ul of PI staining reagent to each well, protect from light for 10 minutes, and put on the flow cytometer.

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 symmetrically structured H3S02.

4.6 Cytokine detection

Take the culture supernatant from the above culture system that has been killed for 48 hours, and use the cytokine detection kit 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), Human Granzyme B ELISABASIC kit (HRP) (mabtech, 3486-1H-20), operate according to the kit instructions , the detection results are shown in Figure 15-16:

Results: During the process of D1-TW mediated killing of two tumor cell lines, 8226 and HCT-8, the cytokines released were close to or lower than those of the symmetrically structured H3S02.

Claims

An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion;

wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is any one of SEQ ID NO: 4, 24, 32 and 40 The amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO: 6, 26, 34 and 42 Item 1, and/or the CD3 binding portion includes 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 such as SEQ ID NO: 1, 27, As shown in any one of 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NO:2, 28, 36 and 44; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3, 29, 37 and 45, wherein the CD3 binding moiety is capable of inducing T cell activation.
The fusion protein of claim 1, wherein the CD47 binding portion includes an anti-CD47 antibody or an antigen-binding fragment thereof, or a CD47 ligand or a functional variant thereof.
The fusion protein of claim 2, wherein the CD47 ligand includes integrin, platelet agglutinin-1, SIRPγ, SIRPα or functional variants or fragments thereof.
The fusion protein of any one of claims 1-3, wherein the CD47 binding portion includes SIRPα, or a functional variant thereof, or a fragment thereof.
An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion;

wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with heavy chain variable regions HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is any one of SEQ ID NO: 4, 24, 32 and 40 The amino acid sequence of the HCDR2 is shown in any one of SEQ ID NO: 5, 25, 33 and 41; the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NO: 6, 26, 34 and 42 Item 1, and/or the CD3 binding portion includes 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 such as SEQ ID NO: 1, 27, As shown in any one of 35 and 43; the amino acid sequence of the LCDR2 is as shown in any one of SEQ ID NO:2, 28, 36 and 44; the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3, 29, As shown in any one of 37 and 45, wherein the CD3 binding portion can induce T cell activation;

Wherein the CD3 binding portion comprises an amino acid sequence having at least 95% identity with the SIRPα protein, the amino acid sequence of the SIRPα protein is shown in SEQ ID NO: 46.
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, said reference antigen binding The protein includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), where HCDR1 contains the amino acid sequence shown in SEQ ID NO:4; where HCDR2 contains The amino acid sequence shown in SEQ ID NO:5; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:6, wherein LCDR1 includes the amino acid sequence shown in SEQ ID NO:1, and LCDR2 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in SEQ ID NO:3, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:3; and

The CD47 binding part competes with the SIRPα protein for binding to human CD47, and the SIRPα protein includes the amino acid sequence shown in SEQ ID NO: 46.
An isolated fusion protein comprising a CD3 binding portion and a CD47 binding portion, wherein the CD3 binding portion includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3 ), wherein HCDR1 includes the amino acid sequence shown in any one of SEQ ID NO:4, 24, 32 and 40; wherein HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO:5, 25, 33 and 41 ; wherein HCDR3 includes the amino acid sequence shown in any one of SEQ ID NO:6, 26, 34 and 42, and wherein LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO:1, 27, 35 and 43 , wherein LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO:2, 28, 36 and 44, and wherein LCDR3 includes the amino acid sequence shown in any one of SEQ ID NO:3, 29, 37 and 45 ;and

The CD47 binding part includes the amino acid sequence shown in SEQ ID NO: 46.
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 HCDR1 The amino acid sequence is as shown in SEQ ID NO:4, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:5 and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:6; and/or the amino acid sequence of the LCDR1 As shown in SEQ ID NO:1, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:2 and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3;

Or; wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO:24; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:25; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:26, wherein LCDR1 includes An amino acid sequence as shown in SEQ ID NO:27, wherein LCDR2 includes an amino acid sequence as shown in SEQ ID NO:28, and wherein LCDR3 includes an amino acid sequence as shown in SEQ ID NO:29;

Or; wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO:32; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:33; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:34, wherein LCDR1 includes An amino acid sequence as shown in SEQ ID NO:35, wherein LCDR2 includes an amino acid sequence as shown in SEQ ID NO:36, and wherein LCDR3 includes an amino acid sequence as shown in SEQ ID NO:37;

Or; wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:42, wherein LCDR1 includes An amino acid sequence as shown in SEQ ID NO:43, wherein LCDR2 includes an amino acid sequence as shown in SEQ ID NO:44, and wherein LCDR3 includes an amino acid sequence as shown in SEQ ID NO:45.
The fusion protein according to any one of claims 1-8, wherein the CD3 binding portion comprises a heavy chain variable region, and the amino acid sequence of the heavy chain variable region is such as SEQ ID NO: 8, 15, 16 , shown in any one of 22, 30 and 38; and/or the CD3 binding part includes a light chain variable region, and the amino acid sequence of the light chain variable region is such as SEQ ID NO: 7, 19, 23, Either one of 31 and 39 is shown.
The fusion protein according to any one of claims 1-9, wherein the CD3 binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO:8 The amino acid sequence shown is, the light chain variable region includes the amino acid sequence shown in SEQ ID NO:7;

Or; 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;

Or; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:19;

Or; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:23;

Or; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:31;

Or; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:39.
According to the fusion protein of any one of claims 1-10, 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.
The fusion protein according to any one of claims 11 to 12, wherein the antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
The fusion protein of any one of claims 1-13, further comprising a heterodimeric Fc portion.
The fusion protein of claim 14, wherein the Fc is from an Fc of IgGl, IgG2, IgG3 or IgG4.
According to the fusion protein of claim 15, the heterodimer Fc portion is connected by a disulfide bond in the hinge region and a knob-in-hole structure of the CH3 domain.
The fusion protein of any one of claims 1-16, 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 IgGl, IgG2, IgG3 or IgG4. district.
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.
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.
The fusion protein of claim 19, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 20.
The fusion protein according to any one of claims 1-20, 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, The antibody light chain contains the amino acid sequence shown in SEQ ID NO:21.
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 IgGl, IgG2, IgG3 or IgG4. district.
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.
The fusion protein according to 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.
A fusion protein having two heavy chains and one light chain, wherein the first heavy chain has VH-CH1-hinge region-Fc from the N-terminus to the C-terminus, and the first light chain has VL- from the N-terminus to the C-terminus. CL; the second heavy chain has SIRPα-hinge region-Fc from the N-terminus to the 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 to bind CD3, SIRPα of the second heavy chain binds CD47;

The first heavy chain includes three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3), and the first light chain includes three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3), among which HCDR1 includes SEQ ID NO:4 , the amino acid sequence shown in any one of 33, 41 and 49; wherein HCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 5, 34, 42 and 50; wherein HCDR3 includes such as SEQ ID NO: 6, The amino acid sequence shown in any one of 35, 43 and 51, wherein LCDR1 includes the amino acid sequence shown in any one of SEQ ID NO: 1, 36, 44 and 52, wherein LCDR2 includes the amino acid sequence shown in any one of SEQ ID NO: 2, The amino acid sequence shown in any one of 37, 45 and 53, and wherein LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 3, 38, 46 and 54; and wherein the second heavy chain includes SIRPα, Wherein the SIRPα includes the amino acid sequence shown in SEQ ID NO:46.
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 HCDR1 The amino acid sequence is as shown in SEQ ID NO:4, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:5 and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:6; and/or the amino acid sequence of the LCDR1 As shown in SEQ ID NO:1, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:2 and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:3;

Or; wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO:24; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:25; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:26, wherein LCDR1 includes An amino acid sequence as shown in SEQ ID NO:27, wherein LCDR2 includes an amino acid sequence as shown in SEQ ID NO:28, and wherein LCDR3 includes an amino acid sequence as shown in SEQ ID NO:29;

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

Or; wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO:40; wherein HCDR2 includes the amino acid sequence shown in SEQ ID NO:41; wherein HCDR3 includes the amino acid sequence shown in SEQ ID NO:42, wherein LCDR1 includes An amino acid sequence as shown in SEQ ID NO:43, wherein LCDR2 includes an amino acid sequence as shown in SEQ ID NO:44, and wherein LCDR3 includes an amino acid sequence as shown in SEQ ID NO:45.
The fusion protein according to 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 such as SEQ ID NO: 8, 15, As shown in any one of 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 such as SEQ ID NO: 7, 19, Any one of 23, 31 and 39 is shown.
The fusion protein of any one of claims 25-27, 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 The region includes the amino acid sequence shown in SEQ ID NO:8, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:7;

Or; 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;

Or; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:19;

Or; 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 includes the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:31; or

Or; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:39.
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.
The fusion protein according to any one of claims 25-30, wherein the first heavy chain and the second heavy chain comprise Fc from human IgG.
The fusion protein of claim 31, wherein the Fc is an Fc from human IgG1, human IgG2, human IgG3 or human IgG4.
The fusion protein according to any one of claims 25 to 32, wherein the first heavy chain and the second heavy chain are connected through a disulfide bond in the hinge region and a pestle structure of the CH3 domain.
The fusion protein of any one of claims 25-33, wherein the first heavy chain and the second heavy chain are of 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 T366W heavy chain substitution, wherein the residues are numbered according to the EU index.
The fusion protein of any one of claims 25-34, wherein the first light chain comprises CL from a human lambda or kappa light chain.
The fusion protein according to any one of claims 25-35, wherein the first heavy chain includes the amino acid sequence shown in SEQ ID NO:18, and the first light chain includes the amino acid sequence shown in SEQ ID NO:21 .
The fusion protein according to any one of claims 25-36, wherein the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 48.
An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-37.
A vector comprising the nucleic acid according to claim 38.
A host cell comprising the expression vector according to claim 39.
A pharmaceutical composition comprising the fusion protein according to any one of claims 1-37 and a pharmaceutically acceptable carrier.
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 method of claim 41 Use of a pharmaceutical composition for the preparation of a medicament for the treatment of cancer.
The use of claim 42, wherein the cancer includes solid tumors and hematological tumors.
The use of claim 42, wherein the cancer is a CD47-expressing cancer.
The use of claim 42, wherein the CD47-expressing cancer is selected from the group consisting of at least one of breast cancer, melanoma, and colon cancer.
A method for 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 the growth of the cancer in the subject.
The method of claim 46, wherein the cancer includes solid tumors and hematological tumors.
The method of claim 46, wherein the cancer is a CD47-expressing cancer.
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.
The method of any one of claims 46-49, further comprising administering a second therapeutic agent to the subject.
The method of claim 50, wherein the second therapeutic agent is an antineoplastic agent, radiation therapy, an antibody drug conjugate, a checkpoint inhibitor, or a combination thereof.
A method for producing the fusion protein of any one of claims 1-37, wherein the method includes culturing the host cell of claim 40 under conditions capable of expressing the fusion protein.
The method of claim 52, wherein the host cell is selected from the group consisting of bacterial cells, fungal cells, plant cells, mammalian cells or viruses;

The method of claim 52, wherein the bacterial cell is E. coli; wherein the fungal cell is a yeast cell;

Wherein the mammalian cells are selected from CHO, NSO, BHK or HEK293 cells.
The method of claim 52, wherein said cells are hybridoma cells.
The method according to claim 54, wherein the hybridoma cells are selected from mouse, rat or rabbit.