IL10 monomer fusion protein and application thereof

Abstract

The invention provides an IL10 monomer fusion protein and application thereof, and relates to the field of biological medicine, and the fusion protein comprises an antibody and an IL-10 monomer. The fusion protein disclosed by the invention has relatively strong targeting property, the safety is remarkably improved, and various tumors or inflammatory diseases can be effectively treated or prevented.

Description

[0001] This application is a divisional application of the invention patent application with application date of November 1, 2022, application number 202211358544.6, and invention name “IL10 monomer fusion protein and its application”.

[0002] Priority declaration

[0003] This application claims priority to Chinese invention patent application number 202111286063.4, filing date November 2, 2021, entitled IL10 monomer fusion protein and its application, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present invention relates to the field of biomedicine, specifically, to a fusion protein and its application, and more specifically, to a fusion protein, a nucleic acid, an expression vector, a recombinant cell, the use of the fusion protein or nucleic acid or expression vector or recombinant cell in the preparation of a drug, and a pharmaceutical composition. Background Art

[0005] Interleukin 10 (IL-10, or IL10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine and a homodimeric secretion. Currently, there are safety issues with clinical IL-10 protein. A small amount of naturally dimerized IL-10 molecules bind to its receptor IL-10Rα with high affinity, and then further bind to IL-10Rβ to form a hexameric complex and activate downstream signals, causing biological functional responses.

[0006] The current clinical IL-10 drug molecules lack targeting, and are prone to off-target and side effects; while most of the IL-10 antibody fusion proteins under development are designed to use the antibody targeting effect to introduce IL-10 to the local environment to exert its effect. These fusion protein molecules often have a normal IL-10 dimer structure, but due to the high affinity binding of IL-10 dimers to IL-10Rα, the targeting effect of the antibody end may be affected, resulting in the failure of the design idea, and if the active dose required by the antibody end is large, the side effects of the IL-10 end are still a thorny problem.

[0007] Therefore, it is still necessary to actively explore IL-10 drugs with strong targeting and few side effects. This research direction is of great significance for the prevention or treatment of tumors or inflammatory diseases. Summary of the invention

[0008] This application is based on the inventor's discovery and understanding of the following problems:

[0009] At present, clinical IL-10 drugs lack targeting, and drug molecules are easily off-target, which not only makes it difficult to exert the efficacy but also easily produces side effects. The inventors explored the connection method between IL-10 monomer molecules and antibody molecules and prepared a fusion protein comprising IL-10 monomer molecules and antibody molecules. Before the antibody binds to the target cell, the IL-10 molecule in the fusion protein is in monomer form and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the antigen on the target cell. After the antibody binds to the antigen, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers and bind to the IL-10 receptor, thereby causing a normal biological response, so that the targeting and safety of the IL-10 drug are significantly improved, and the efficacy is fully exerted.

[0010] In the first aspect of the present invention, the present invention proposes a fusion protein. According to an embodiment of the present invention, it includes an antibody and an IL-10 monomer, wherein the antibody includes two identical light chains and two identical heavy chains, an IL-10 monomer is connected to the antibody heavy chain or light chain, and the N-terminus of the IL-10 monomer is not connected to the C-terminus of the antibody heavy chain. The natural IL-10 molecule is in the form of a dimer. According to the fusion protein of the embodiment of the present invention, the IL-10 molecule is in the form of a monomer before the antibody binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell. After the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers, bind to the IL-10 receptor, thereby causing a normal biological response, and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the dosage limit of the natural dimer IL-10 molecule or its antibody fusion protein, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0011] According to an embodiment of the present invention, the above fusion protein may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, each antibody light chain includes a VL region and a CL region, and each antibody heavy chain includes a VH region, a CH1 region, a CH2 region, and a CH3 region; the N-terminus of the IL-10 monomer is connected to the C-terminus of the antibody light chain, or the C-terminus of the IL-10 monomer is connected to the N-terminus of the antibody light chain, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the hydrogen chain VL region, the C-terminus of the IL-10 monomer is connected to the N-terminus of the hydrogen chain CL region, or the C-terminus of the IL-10 monomer is connected to the N-terminus of the antibody heavy chain, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain of the antibody, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain VH region, the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH1 region, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH3 region.

[0013] According to an embodiment of the present invention, the IL-10 monomer comprises a natural IL-10 monomer.

[0014] According to an embodiment of the present invention, the IL-10 monomer is a modified natural IL-10 monomer, and the modification includes at least one of the following (i) and (ii): (i) removing the first two amino acids at the N-terminus of the natural IL-10 monomer; (ii) inserting a first connecting peptide between amino acids at positions 116 and 117, wherein the first connecting peptide is a flexible connecting peptide.

[0015] After removing the first two amino acids at the N-terminus of the natural IL-10 monomer, the stability of the IL-10 monomer is improved. At the same time, after inserting the first connecting peptide between the 116th and 117th amino acids of the IL-10 monomer, the IL-10 monomer molecule in the fusion protein is closed to become a closed-loop IL-10 monomer molecule (the two IL10 chains of the natural IL10 dimer are staggered together. After the linker is inserted, the IL10 monomer can independently form a structural domain, that is, a closed-loop monomer molecule). The two IL-10 monomers in the same fusion protein will not be connected to each other or interfere with each other.

[0016] According to an embodiment of the present invention, the amino acid sequence of the first connecting peptide is shown as SEQ ID NO:21.

[0017] GGGSGG (SEQ ID NO: 21)

[0018] According to an embodiment of the present invention, the IL-10 monomer has the amino acid sequence shown in SEQ ID NO:2.

[0019] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNA FNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 2).

[0020] According to an embodiment of the present invention, the IL-10 monomer is connected to the antibody heavy chain or light chain via a second connecting peptide or / and a third connecting peptide. After the IL10 monomer is connected to the antibody heavy chain or light chain using the second connecting peptide or / and the third connecting peptide, after the antibody portion in the fusion protein binds to the corresponding target cell, the IL-10 monomers of the two fusion proteins that are close to each other are more likely to form dimers to exert their effects and bind to the IL-10 receptor.

[0021] According to an embodiment of the present invention, the second connecting peptide and / or the third connecting peptide may be a flexible connecting peptide or a rigid connecting peptide.

[0022] According to an embodiment of the present invention, the second connecting peptide has an amino acid sequence shown in SEQ ID NO:12, and the third connecting peptide has an amino acid sequence shown in SEQ ID NO:22.

[0023] GSGSGSGS (SEQ ID NO: 12).

[0024] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 22).

[0025] According to an embodiment of the present invention, the IL-10 monomer is connected to the heavy chain end or the light chain end of the antibody via the second connecting peptide.

[0026] According to an embodiment of the present invention, the N-terminus of the second connecting peptide is connected to the C-terminus of the antibody light chain, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody heavy chain.

[0027] According to an embodiment of the present invention, the IL-10 monomer is connected to the heavy chain end or the light chain end of the antibody via the third connecting peptide;

[0028] According to an embodiment of the present invention, the N-terminus of the third connecting peptide is connected to the C-terminus of the antibody light chain, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the antibody heavy chain, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the antibody heavy chain.

[0029] According to an embodiment of the present invention, the N-terminus of the third connecting peptide is connected to the C-terminus of the light chain VL region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the light chain CL region; or the N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain VH region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain CH1 region; or the N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain CH3 region.

[0030] According to an embodiment of the present invention, the N-terminus of the IL-10 monomer is connected to the C-terminus of the VL region of the light chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CL region of the light chain of the antibody through the second connecting peptide; or the N-terminus of the IL-10 monomer is connected to the C-terminus of the VH region of the heavy chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CH1 region of the heavy chain of the antibody through the second connecting peptide; or the N-terminus of the IL-10 monomer is connected to the C-terminus of the CH2 region of the heavy chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CH3 region of the heavy chain of the antibody through the second connecting peptide.

[0031] According to an embodiment of the present invention, the antibody is selected from immune cell antibodies and tumor cell antibodies.

[0032] According to an embodiment of the present invention, the antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Her2 antibody, an anti-CCR8 antibody, an anti-VEGFR2 antibody, an anti-claudin18.2 antibody, an anti-CD39 antibody, an anti-SMA4D antibody, an anti-GUCY2C antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-CD47 antibody or an anti-TNFα antibody.

[0033] According to an embodiment of the present invention, the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:9; or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:5; or the fusion protein has the amino acid sequence described in SEQ ID NO:4 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:7 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:8; or the fusion protein has the amino acid sequence described in SEQ ID NO:10 and SEQ ID NO:6.

[0034] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3).

[0035] DTVLTQSPALAVSLGQRVTISCKASETVSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSGGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVK TFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO:9).

[0036] DTVLTQSPALAVSLGQVRTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRGSGSGSGSGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQD PDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGSGSGSGSTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:5).

[0037] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSGSGSGSGSGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGSGSGSGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。

[0038] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:6)。

[0039] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGSGSGSGSGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGSGSGSGSGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7)。

[0040] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSGGGSGGGSGGGSGDTVLTQSPALAVSLGQRVTISCKASETVSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:8).

[0041] (SEQ ID NO:10).

[0042] In the second aspect of the present invention, the present invention proposes a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the fusion protein described in the first aspect. The IL-10 molecule in the fusion protein encoded by the nucleic acid according to the embodiment of the present invention is in a monomeric form before the antibody part binds to the target cell, cannot bind to its receptor, and does not interfere with the binding of the antibody molecule to the target cell; after the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers and bind to the IL-10 receptor, thereby causing a normal biological response; and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the natural dimer IL-10 molecule or its antibody fusion protein dosage limit, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0043] According to an embodiment of the present invention, the nucleic acid molecule is DNA or RNA.

[0044] In the third aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, the expression vector comprises the nucleic acid described in the second aspect. After the expression vector according to the embodiment of the present invention is introduced into a suitable receptor cell, the expression of the fusion protein described above can be effectively realized under the mediation of the regulatory system, and the IL-10 molecule in the obtained fusion protein is in a monomeric form before the antibody part binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; after the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers, bind to the IL-10 receptor, and thus cause a normal biological response; and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the natural dimer IL-10 molecule or its antibody fusion protein dosage limit, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0045] In the fourth aspect of the present invention, the present invention proposes a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries the nucleic acid described in the second aspect, the expression vector described in the third aspect, or expresses the fusion protein described in the first aspect. The recombinant cell according to the embodiment of the present invention can be used for the expression and large-scale acquisition of the fusion protein described above. The obtained fusion protein is in the form of a monomer before the antibody part binds to the target cell, and the IL-10 molecule cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; after the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers, bind to the IL-10 receptor, and thus cause a normal biological reaction; and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the dosage limit of the natural dimer IL-10 molecule or its antibody fusion protein, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0046] In the fifth aspect of the present invention, the present invention proposes the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of a drug. According to an embodiment of the present invention, the drug is used to treat or prevent tumors or inflammatory diseases. According to a specific embodiment of the present invention, the drug can effectively treat or alleviate tumors and inflammatory diseases, and has high safety.

[0047] In a sixth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the composition comprises the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect. The medicine according to a specific embodiment of the present invention can effectively treat or alleviate tumors and inflammatory diseases and has high safety.

[0048] According to an embodiment of the present invention, the above-mentioned pharmaceutical composition may further include at least one of the following additional technical features (i) and (ii):

[0049] (i) According to an embodiment of the present invention, the pharmaceutical composition is used for treating or preventing tumors or inflammatory diseases.

[0050] (ii) According to an embodiment of the present invention, the pharmaceutical composition may include: a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient includes at least one of a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent; and the pharmaceutical composition is in the form of at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, and a lyophilized preparation.

[0051] In the seventh aspect of the present invention, the present invention proposes a method for diagnosing, treating, preventing or alleviating a disease, disorder or condition associated with a tumor or inflammatory disease in a subject, which comprises administering a therapeutically effective amount of the aforementioned fusion protein and / or the aforementioned pharmaceutical composition to the above-mentioned subject.

[0052] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which:

[0054] Figure 1 Schematic diagram of the binding mode of natural IL-10 molecules and IL-10 monomers to IL-10 receptors according to an embodiment of the present invention, wherein A represents a schematic diagram of the binding mode of natural IL-10 molecules to IL-10 receptors, R1 represents IL-10 receptor (IL-10Rα), and R2 represents IL-10 receptor (IL-10Rβ); B represents a schematic diagram of the binding mode of IL-10 monomers to IL-10 receptors, R1 represents IL-10 receptor (IL-10Rα), and IL-10M1 represents IL-10 monomers;

[0055] Figure 2 Schematic diagram of the binding of the antibody in the fusion protein according to an embodiment of the present invention to the antigen on the target cell and the binding of the IL-10 monomer to the IL-10 receptor. The affinity of the IL-10 monomer is weak and the targeting effect is poor. Therefore, the antibody end can play a normal targeting role. After targeting the tumor surface, the local enrichment of the antibody can aggregate the IL-10 monomers together to form a dimer IL-10(M)*2 to play a role. Among them, IL-10R represents the IL10RαRβ complex, Her2 represents the human epidermal growth factor receptor, which is an example of the antibody targeting antigen, T-cell represents T cell, and Tumor represents tumor;

[0056] Figure 3: is a schematic diagram of the position structure of the IL-10 monomer inserted into the antibody molecule according to an embodiment of the present invention, wherein A represents the position (position 1-position 7) where the IL-10 monomer can be inserted on the antibody, B represents a schematic diagram of the structure of inserting an IL-10 monomer between the VL and CL of each light chain of the antibody (i.e., position 1), and connecting them using linker2, and the resulting fusion protein molecule is named R0987; C represents a schematic diagram of the structure of inserting an IL-10 monomer between the VH and CH1 of the heavy chain of the antibody (i.e., position 2), and connecting them using linker2, and the resulting fusion protein molecule is named R0988; D represents a schematic diagram of the structure of inserting an IL-10 monomer between the CH2 and CH3 of the heavy chain of the antibody (i.e., position 3), and connecting them using linker2, and the resulting fusion protein molecule is named R0989; E represents a schematic diagram of inserting an IL-10 monomer at the N-terminus (i.e., position 4) of the light chain of the antibody The schematic diagram of the structure of an IL-10 monomer is connected by linker 2, and the resulting fusion protein molecule is named R0990; F is a schematic diagram of the structure of an IL-10 monomer inserted at the C-terminus (i.e., position 5) of the antibody light chain, and connected by linker 3, and the resulting fusion protein molecule is named R0991; G represents a schematic diagram of the structure of an IL-10 monomer inserted at the N-terminus (i.e., position 6) of the antibody heavy chain, and connected by linker 3, and the resulting fusion protein molecule is named R0992; H represents a schematic diagram of the structure of an IL-10 monomer inserted at the C-terminus (i.e., position 7) of the antibody heavy chain, and connected by linker 3, and the resulting fusion protein molecule is named R0993; I is a structural diagram of the control molecules (R0428, R0594, R0862, R0676, R0579, R1049) in Examples 3-5 of the present invention;

[0057] Figure 4 This is a diagram showing the SDS-PAGE detection results of fusion proteins R0987, R0988, R0989, R0990, R0991, R0992, and R0993 according to an embodiment of the present invention;

[0058] Figure 5 : is a result diagram of detecting antibody end binding activity by flow cytometry according to the fusion protein of an embodiment of the present invention, wherein the abscissa (Ab conc.Log (nM)) represents the antibody concentration (nM), and the ordinate MFIPE represents the mean fluorescence intensity;

[0059] Figure 6 This is a result diagram of detecting the IL-10 monomer end binding activity by flow cytometry according to the fusion protein of an embodiment of the present invention, wherein the abscissa (Ab conc.Log (nM)) represents the antibody concentration (nM), and the ordinate MFIPE represents the mean fluorescence intensity;

[0060] Figure 7This is a result diagram of detecting the IL-10 monomer end binding activity using a reporter gene method for the fusion protein according to an embodiment of the present invention, wherein the abscissa (Ab conc.Log (μg / mL)) represents the antibody concentration (μg / mL), and the ordinate (Lum) represents the luminescence fluorescence intensity;

[0061] Figure 8 This is a statistical analysis result diagram of the tumor volume in mice after the MC38 tumor model mice were treated with the fusion protein according to an embodiment of the present invention, wherein the abscissa (Days post engraftment) represents the number of days after the mice were transplanted, and the ordinate (Tumor volume) represents the tumor volume;

[0062] Fig. 9 This is a statistical analysis result diagram of the tumor volume in mice after the CT26 tumor model mice were treated with the fusion protein according to an embodiment of the present invention, wherein the horizontal axis (Days post engraftment) represents the number of days after the mouse transplantation, and the vertical axis (Tumor volume) represents the tumor volume. DETAILED DESCRIPTION

[0063] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0064] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0065] As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to an antigen. It includes two light chains with a relatively low molecular weight and two heavy chains with a relatively high molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino acid sequence at the amino end (N end) of the peptide chain varies greatly, which is called the variable region (V region), while the carboxyl end (C end) is relatively stable and varies little, which is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively, the C region of the L chain is CL, and the C region of the H chain includes the CH1 region, the CH2 region, and the CH3 region.

[0066] The natural IL-10 molecule is a dimer structure (including two natural IL-10 monomers). The natural IL-10 molecule binds to the IL-10 receptor (IL-10R) in the following way: Figure 1-A, while the activity of IL-10 monomer on IL-10Rα is 10 times weaker than that of natural IL-10 dimer (specific experimental data reference: J Biol Chem. 2000 May 5; 275 (18): 13552-7. doi: 10.1074 / jbc.275.18.13552.), and it is almost unable to mediate further binding with IL-10Rβ, so it is difficult to activate downstream signals to cause biological functional responses. The binding mode of IL-10 monomer to IL-10 receptor is as follows Figure 1 -B.

[0067] Therefore, the present invention connects IL-10 monomers to different parts of the antibody molecule (which can be symmetrical or asymmetrical structures) to construct the fusion protein (IL-10 monomer fusion protein), and the IL-10 monomers are isolated by the steric hindrance of the antibody structure itself. When the IL-10 monomer fusion protein is in a free state, even if the IL-10 monomer binds to IL-10Rα, it cannot cause a functional response. Only when the antibody end binds to the target cell can it gather the adjacent IL-10 monomer-IL-10Rα complex through the aggregation effect, and further bind to IL-10Rβ to form a complex to generate downstream signals and trigger biological functions. The specific binding method is as follows Figure 2 As shown, this can avoid the interference of IL-10 molecules on the targeting of the antibody before the drug molecules reach the target site, and will not produce clinical side effects; and because IL-10 monomers are difficult to induce biological functions, the IL-10 monomer fusion protein will not produce the dosage limit of the natural dimer IL-10 molecule or its antibody fusion protein, so that the dosage of the IL-10 monomer fusion protein is significantly improved. At the same time, since the clinical use doses of different antibodies are different, when the use dose of IL-10 molecules is low, it will seriously limit the selection of the antibody target of the IL-10 monomer fusion protein. Therefore, the fusion protein obtained by the present invention provides a wider space for the fused antibody end to function, so that the IL-10 monomer fusion protein can be used with more antibody molecules.

[0068] In some embodiments, the present invention proposes a fusion protein, including an antibody and an IL-10 monomer, wherein the antibody includes two identical light chains and two identical heavy chains, an IL-10 monomer is connected to the antibody heavy chain or light chain, and the N-terminus of the IL-10 monomer is not connected to the C-terminus of the antibody heavy chain. The natural IL-10 molecule is in the form of a dimer. According to some specific embodiments of the present invention, the IL-10 molecule is in the form of a monomer before the antibody binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell. After the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers and bind to the IL-10 receptor, thereby causing a normal biological response, so that the targeting and safety of the fusion protein are significantly improved, and the efficacy is fully exerted.

[0069] According to some specific embodiments of the present invention, each antibody light chain includes a VL region and a CL region, and each antibody heavy chain includes a VH region, a CH1 region, a CH2 region, and a CH3 region; the N-terminus of the IL-10 monomer is connected to the C-terminus of the antibody light chain, or the C-terminus of the IL-10 monomer is connected to the N-terminus of the antibody light chain, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the hydrogen chain VL region, the C-terminus of the IL-10 monomer is connected to the N-terminus of the hydrogen chain CL region, or the C-terminus of the IL-10 monomer is connected to the N-terminus of the antibody heavy chain, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain VH region, the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH1 region, or the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH3 region.

[0070] According to some specific embodiments of the present invention, the IL-10 monomer includes a natural IL-10 monomer, and the natural IL-10 monomer has the amino acid sequence shown in SEQ ID NO:1.

[0071] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:1)

[0072] According to some specific embodiments of the present invention, the IL-10 monomer includes a modified natural IL-10 monomer, and the modification includes at least one of the following (i) and (ii): (i) removing the first two amino acids at the N-terminus of the natural IL-10 monomer; (ii) inserting a first connecting peptide between amino acids at positions 116 and 117, wherein the first connecting peptide is a flexible connecting peptide.

[0073] After removing the first two amino acids at the N-terminus of the natural IL-10 monomer, the stability of the IL-10 monomer is improved. At the same time, after inserting the first connecting peptide between the 116th and 117th amino acids of the IL-10 monomer, the IL-10 monomer molecule in the fusion protein becomes a closed-loop monomer molecule (the two IL10 chains of the natural IL10 dimer are staggered together. After the linker is inserted, the IL10 monomer can independently form a structural domain, that is, a closed-loop monomer molecule), and the two IL-10 monomers in the same fusion protein will not be connected to each other or interfere with each other.

[0074] According to some specific embodiments of the present invention, the first connecting peptide has an amino acid sequence shown in SEQ ID NO:21.

[0075] GGGSGG (SEQ ID NO: 21)

[0076] According to some specific embodiments of the present invention, the IL-10 monomer has the amino acid sequence shown in SEQ ID NO:2.

[0077] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNA FNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 2).

[0078] According to an embodiment of the present invention, the IL-10 monomer is connected to the antibody heavy chain or light chain via a second connecting peptide or / and a third connecting peptide. After the IL10 monomer is connected to the antibody heavy chain or light chain using the second connecting peptide or / and the third connecting peptide, after the antibody portion in the fusion protein binds to the corresponding target cell, the IL-10 monomers of the two fusion proteins that are close to each other are more likely to form dimers to exert their effects and bind to the IL-10 receptor.

[0079] According to an embodiment of the present invention, the second connecting peptide and / or the third connecting peptide are not particularly limited, and may be either a flexible connecting peptide or a rigid connecting peptide.

[0080] According to an embodiment of the present invention, the second connecting peptide has an amino acid sequence shown in SEQ ID NO:12, and the third connecting peptide has an amino acid sequence shown in SEQ ID NO:22.

[0081] GSGSGSGS (SEQ ID NO: 12).

[0082] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 22).

[0083] According to an embodiment of the present invention, the IL-10 monomer is connected to the heavy chain end or the light chain end of the antibody via the second connecting peptide.

[0084] According to an embodiment of the present invention, the N-terminus of the second connecting peptide is connected to the C-terminus of the antibody light chain, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the second connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the antibody heavy chain.

[0085] According to an embodiment of the present invention, the IL-10 monomer is connected to the heavy chain end or the light chain end of the antibody via the third connecting peptide;

[0086] According to an embodiment of the present invention, the N-terminus of the third connecting peptide is connected to the C-terminus of the antibody light chain, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the antibody light chain; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the antibody heavy chain.

[0087] According to an embodiment of the present invention, the N-terminus of the third connecting peptide is connected to the C-terminus of the light chain VL region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the light chain CL region; or the N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain VH region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain CH1 region; or the N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or the N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain CH3 region.

[0088] According to an embodiment of the present invention, the N-terminus of the IL-10 monomer is connected to the C-terminus of the VL region of the light chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CL region of the light chain of the antibody through the second connecting peptide; or the N-terminus of the IL-10 monomer is connected to the C-terminus of the VH region of the heavy chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CH1 region of the heavy chain of the antibody through the second connecting peptide; or the N-terminus of the IL-10 monomer is connected to the C-terminus of the CH2 region of the heavy chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CH3 region of the heavy chain of the antibody through the second connecting peptide.

[0089] According to some specific embodiments of the present invention, the antibody is selected from immune cell antibodies and tumor cell antibodies.

[0090] According to some specific embodiments of the present invention, the antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Her2 antibody, an anti-CCR8 antibody, an anti-VEGFR2 antibody, an anti-claudin18.2 antibody, an anti-CD39 antibody, an anti-SMA4D antibody, an anti-GUCY2C antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-CD47 antibody or an anti-TNFα antibody.

[0091] According to some specific embodiments of the present invention, the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:9; or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQ ID NO:5; or the fusion protein has the amino acid sequence described in SEQ ID NO:4 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:7 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:3 and SEQID NO:8; or the fusion protein has the amino acid sequence described in SEQ ID NO:11 and SEQ ID NO:6; or the fusion protein has the amino acid sequence described in SEQ ID NO:10 and SEQ ID NO:6.

[0092] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALK SRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:3).

[0093] DTVLTQSPALAVSLGQRVTISCKASETVSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSGGGGSGGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO:9).

[0094] DTVLTQSPALAVSLGQVRTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRGSGSGSGSGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQD PDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGSGSGSGSTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:5).

[0095] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSGSGSGSGSGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGSGSGSGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:4)。

[0096] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:6)。

[0097] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGSGSGSGSGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGSGSGSGSGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7)。

[0098] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSGGGSGGGSGGGSGDTVLTQSPALAVSLGQRVTISCKASETVSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:8).

[0099] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGGSGGGGSGGGGSGGGGSGQVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10)。

[0100] (SEQ ID NO:11)

[0101] DTVLTQSPALAVSLGQVRTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:6)

[0102] In some embodiments, the present invention proposes an isolated nucleic acid encoding the fusion protein described in the first aspect. According to the fusion protein encoded by the nucleic acid in some specific embodiments of the present invention, the IL-10 molecule is in monomeric form before the antibody part binds to the target cell, and cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; after the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers and bind to the IL-10 receptor, thereby causing a normal biological response; and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the dosage limit of the natural dimer IL-10 molecule or its antibody fusion protein, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0103] According to some specific embodiments of the present invention, the nucleic acid molecule is DNA or RNA.

[0104] In some embodiments, the present invention proposes an expression vector, which comprises the nucleic acid described in the second aspect. When the above-mentioned nucleic acid molecule is connected to the vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule can be operably connected to the control element. In this article, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their intended function of regulating the transcription and translation of the exogenous gene. After the expression vector in some specific embodiments of the present invention is introduced into a suitable receptor cell, the expression of the fusion protein described above can be effectively achieved under the mediation of the regulatory system. The obtained fusion protein is in the form of a monomer before the antibody part binds to the target cell, and the IL-10 molecule cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; after the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers and bind to the IL-10 receptor, thereby causing a normal biological reaction; and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the dosage limit of the natural dimer IL-10 molecule or its antibody fusion protein, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0105] According to some specific embodiments of the present invention, the expression vector is a eukaryotic expression vector.

[0106] In some embodiments, the present invention proposes a recombinant cell, which carries the nucleic acid described in the second aspect, the expression vector described in the third aspect, or expresses the fusion protein described in the first aspect. The recombinant cell according to the specific embodiment of the present invention can be used for the expression and large-scale acquisition of the fusion protein described above. The obtained fusion protein is in the form of a monomer before the antibody part binds to the target cell, and the IL-10 molecule cannot bind to its receptor, and will not interfere with the binding of the antibody molecule to the target cell; after the antibody binds to the target cell, a large number of IL-10 monomer molecules aggregate, so that the two IL-10 monomers close to each other act as dimers and bind to the IL-10 receptor, thereby causing a normal biological response; and because the IL-10 monomer is difficult to induce biological function, the IL-10 monomer fusion protein will not produce the dosage limit of the natural dimer IL-10 molecule or its antibody fusion protein, so that the dosage of the IL-10 monomer fusion protein is significantly increased. The fusion protein can be used in combination with more antibody molecules, so that the targeting and safety of the fusion protein are significantly improved, the efficacy is fully exerted, and a variety of tumors or inflammatory diseases can be treated or prevented.

[0107] According to some specific embodiments of the present invention, the recombinant cell is a mammalian cell, such as a human, monkey, rabbit, dog, cow, etc.; a mammalian cell is such as a human HEK-293F cell or a CHO-K1 cell.

[0108] According to some specific embodiments of the present invention, the recombinant cell does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0109] In some embodiments, the present invention provides the use of the above-mentioned fusion protein, nucleic acid, expression vector or recombinant cell in the preparation of a drug for treating or preventing a tumor or inflammatory disease. According to some specific embodiments of the present invention, the drug can effectively treat or alleviate tumors and inflammatory diseases and has high safety.

[0110] In some embodiments, the present invention proposes a pharmaceutical composition comprising the aforementioned fusion protein, nucleic acid, expression vector or recombinant cell. The pharmaceutical composition according to some specific embodiments of the present invention can accurately bind to the target cell to which the antibody contained in the pharmaceutical composition can bind, and the IL-10 monomer molecule in the pharmaceutical composition has a low binding activity with the IL-10 receptor before the antibody and the target cell bind, so the IL-10 monomer will not interfere with the targeting of the antibody contained in the pharmaceutical composition, and fully exerts its efficacy after the antibody contained in the pharmaceutical composition binds to the target cell, effectively treats or alleviates tumors and inflammatory diseases, and has high safety.

[0111] According to some specific embodiments of the present invention, the pharmaceutical composition is used to treat or prevent tumors or inflammatory diseases.

[0112] According to some specific embodiments of the present invention, the fusion protein provided by the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject. Generally, these pharmaceutical compositions include the fusion protein provided by the present invention.

[0113] According to some specific embodiments of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, adhesive, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid adhesive, glidant or lubricant, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the fusion protein of the present invention, such as any adverse biological effect produced or any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0114] For example, the fusion protein of the present invention can be incorporated into a pharmaceutical composition suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersants or suspending agents, tablets, pills, powders, liposomes and suppositories. The fusion protein can be administered by intravenous infusion or intramuscular or subcutaneous injection.

[0115] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art recognizes as having potential benefits or therapeutic effects. Moreover, the treatment or prevention provided by the present invention may include the treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, such as cancer. Additionally, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or conditions.

[0116] According to some specific embodiments of the present invention, the present invention provides a method for diagnosing, treating, preventing or alleviating a disease, disorder or condition associated with a tumor or inflammatory disease in a subject, comprising administering a therapeutically effective amount of the aforementioned fusion protein and / or the aforementioned pharmaceutical composition to the above-mentioned subject.

[0117] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0118] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0119] Example 1 Construction of fusion protein

[0120] In this embodiment, the IL-10 monomer is inserted into different positions of the antibody molecule (this embodiment takes the anti-PD-1 antibody as an example) to obtain a fusion protein, wherein the specific experimental operation of the construction of the IL-10 monomer molecule mainly refers to the "Molecular Cloning Experiment Guide". The natural IL-10 monomer molecule sequence is shown in SEQ ID NO: 1, and a peptide segment (i.e., the first connecting peptide, linker1) with an amino acid sequence as shown in SEQ ID NO: 21 is inserted between the two sites 116N and 117K of the natural IL-10 monomer molecule sequence, and the first two amino acids at the N-terminus of the natural IL-10 monomer are removed, thereby forming the IL-10 monomer of the embodiment of the present invention, whose sequence is shown in SEQ ID NO: 2; the antibody heavy chain is shown in SEQ ID NO: 3, and the antibody light chain is shown in SEQ ID NO: 6.

[0121] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 1).

[0122] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNA FNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 2).

[0123] GGGSGG (SEQ ID NO: 21)

[0124] In this example, a total of 7 fusion proteins were constructed. The specific structures of the fusion proteins are as follows: Figure 3 As shown (the insertion position of the IL-10 monomer is shown in the figure); the connection mode of the IL-10 monomer and the antibody molecule in the fusion protein is described as follows:

[0125] R0987: The IL-10 monomer is inserted between the VL and CL of the antibody light chain (position 1), and is connected using linker 2 (GSGSGSGS). The resulting fusion protein has the amino acid sequence shown in SEQ ID NO: 3 (heavy chain) and SEQ ID NO: 5 (light chain), as shown in Figure 3 As shown in B;

[0126] R0988: The IL-10 monomer is inserted between the VH and CH1 of the antibody heavy chain (position 2), and is connected using linker 2 (GSGSGSGS). The resulting fusion protein has the amino acid sequence shown in SEQ ID NO: 4 (heavy chain) and SEQ ID NO: 6 (light chain), as shown in Figure 3 As shown in C;

[0127] R0989: The IL-10 monomer is inserted between CH2 and CH3 of the antibody heavy chain (position 3), and is connected using linker 2 (GSGSGSGS). The resulting fusion protein has the amino acid sequence shown in SEQ ID NO: 7 (heavy chain) and SEQ ID NO: 6 (light chain), as shown in Figure 3 As shown in D;

[0128] R0990: The IL-10 monomer is connected to the N-terminus (position 4) of the antibody light chain via a linker, and is connected using linker 3 (GGGGSGGGGSGGGGSGGGGSG), and the resulting fusion protein has the amino acid sequence shown in SEQ ID NO: 3 (heavy chain) and SEQ ID NO: 8 (light chain), as shown in Figure 3 As shown in E;

[0129] R0991: The IL-10 monomer was connected to the C-terminus (position 5) of the antibody light chain via a linker, and the fusion protein obtained had the amino acid sequence shown in SEQ ID NO: 3 (heavy chain) and SEQ ID NO: 9 (light chain), as shown in FIG. Figure 3 As shown in F;

[0130] R0992: The IL-10 monomer was connected to the N-terminus (position 6) of the antibody heavy chain via a linker and connected using linker 3 (GGGGSGGGGSGGGGSGGGGSG), and the resulting fusion protein had an amino acid sequence as shown in SEQ ID NO: 10 (heavy chain) and SEQ ID NO: 6 (light chain), as shown in Figure 3 As shown in G;

[0131] R0993: IL-10 monomer was linked to the C-terminus (position 7) of the antibody heavy chain via a linker and used linker3

[0132] (GGGGSGGGGSGGGGSGGGGSG), the resulting fusion protein has the amino acid sequence shown in SEQ ID NO: 11 (heavy chain) and SEQ ID NO: 6 (light chain), as shown in Figure 3 As shown in H;

[0133] The structures of the control molecules (R0428, R0594, R0862, R0676, R0579, R1049) in Examples 3-5 of the present invention are shown in Figure 3 As shown in I.

[0134] The second connecting peptide (linker2) and the third connecting peptide (linker3) are flexible or rigid connecting peptides, wherein the second connecting peptide has an amino acid sequence as shown in SEQ ID NO: 12, and the third connecting peptide has an amino acid sequence as shown in SEQ ID NO: 22;

[0135] The amino acid sequences of the heavy chain and light chain of the above fusion protein are shown in Table 1 below, wherein the sequences marked in bold italics are

[0136] GSGSGSGS (SEQ ID NO: 12) is the amino acid sequence of the second linker peptide (linker 2), only the sequence marked in bold

[0137] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 22) is the amino acid sequence of the third linker peptide (linker 3), and the sequence marked only with underscores

[0138] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALS

[0139] EMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 2) is the amino acid sequence of the engineered IL-10 monomer.

[0140] Table 1

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] Example 2 Preparation of fusion protein

[0147] A plasmid containing the target gene (encoding the fusion protein in Example 1) was constructed and prepared by conventional methods. The plasmid containing the target gene was introduced into the host cell Expi293 after forming a cationic complex with the transfection reagent PEI. During the time when the plasmid was in the cell, the exogenous gene on the plasmid was transcribed and translated in the cell, thereby obtaining the fusion protein. The specific experimental operation is as follows:

[0148] Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm. Before transfection, 2E6 cells were inoculated into a 1L shake flask by cell counting. The culture system was about 300mL. Prepare transfection complexes for transfection: First, add 750μg of target plasmid to a 50mL centrifuge tube containing 15mL Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5mg of transfection reagent PEI to a 50mL centrifuge tube containing 15mL Opti-MEM reagent, mix gently, and incubate at room temperature for 5min, marked as tube B; add the PEI dilution in tube B dropwise to the DNA dilution in tube A, mix gently, and incubate at room temperature for 15min. After the incubation, add the PEI-target plasmid complex to Expi293 cells, place it in a 37°C shaker and continue to culture it. Collect samples after culturing to D5-D10.

[0149] The transient cell expression solution was centrifuged at 9000rpm / 20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. ProA affinity chromatography was used for purification. The process is as follows: Use AKTA avant 150 chromatography equipment, balance the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, so that the target protein is adsorbed on the chromatography column and other impurities penetrate and separate. After the sample is loaded, use at least 5CV equilibration buffer (10mM PBS) to rinse the chromatography column again, and then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein. Neutralization buffer (1M Tris, pH=8.0) is added to the collection tube in advance. The volume of neutralization buffer added is determined according to the estimated content of the eluted sample, and generally 10% of the elution volume is added.

[0150] The expression data of IL-10 monomer fusion protein are shown in Table 2. Except for R0987, which has an expression level of 97.731 mg / mL, the transient expression levels of the other fusion proteins are all greater than 100 mg / L, and the expression level of R0990 even reaches 194.409 mg / L. After one-step affinity purification, the samples were tested by SEC-HPLC, and the target purity was above 85%, and the purity of R0989 was even greater than 95%. The samples were tested by SDS-PAGE, showing the correct distribution of light and heavy chains. The electrophoresis results are as follows Figure 4 It was concluded that the IL-10 monomer fusion proteins at different insertion positions all exhibited good production properties.

[0151] Table 2

[0152]

[0153] Example 3 In vitro activity of fusion protein

[0154] 3.1 Detection of binding activity of fusion protein antibody end and IL-10 monomer end by flow cytometry (FACS)

[0155] All fusion protein molecules, R0428 (positive control antibody without IL-10, whose heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 13 and 14) or R0594 (IL-10-Fc fusion protein control, whose amino acid sequence is shown in SEQ ID NO: 15, wherein IL-10 removes the first two amino acids of natural IL-10 and does not insert the first connecting peptide) protein molecules were diluted to an initial concentration of 400 nM, a volume of 180 μl, and 3-fold gradient dilution (60 μl sample + 120 μl dilution buffer) to obtain a total of 11 concentration gradient points. CHO-mPD1 or CHO-hIL-10R cells were centrifuged at 250g for 5 min, the supernatant was discarded, the cell density was adjusted to 2E+06 with 3% BSA PBS buffer, and 100 μL / tube was evenly distributed into 96-well V-shaped plates; the diluted fusion protein molecules were added to the cells, 100 μL / well, and incubated at 2-8°C for 0.5h; the 96-well plate was removed, centrifuged at 250g for 5 min, the supernatant was carefully removed, 200 μl / well of 3% BSA PBS buffer was added, and centrifuged at 250g for 5 min again, and the supernatant was carefully removed; PE fluorescent secondary antibody (1:500 dilution) was prepared with PBS buffer containing 3% BSA, and added to the corresponding 96-well plate at a volume of 100 μl / well, the cells were resuspended, and incubated at 2-8°C for 30min; the 96-well plate was removed, centrifuged at 250g for 5 min, the supernatant was carefully removed, and PBS buffer containing 3% BSA was added. 200 μl / well, centrifuge again at 250g for 5 min, carefully remove the supernatant; resuspend in 1xPBS at a volume of 100 μL / well, and perform FACS detection after resuspension.

[0156] Heavy chain sequence of R0428:

[0157] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQID NO:13).

[0158] The light chain sequence of R0428:

[0159] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQID NO:14).

[0160] R0594 contains two identical peptide chains. The amino acid sequence of one of the chains:

[0161] GQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIF INYIEAYMTMKIRNGGGSGSGSGSEPKSSDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKQL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:15).

[0162] The antibody end binding activity test results are as follows Figure 5 As shown, the activity of the antibody end of fusion proteins R0987, R0988, R0989, and R0991 is relatively strong, and R0990 is the weakest. This shows that the insertion of IL-10 monomer between the light chain VL and CL, or between the heavy chain VH and CH1, or between the heavy chain CH2 and CH3, the C-terminus of the light chain does not affect the binding activity of the antibody.

[0163] The results of the IL-10 monomer end binding activity test to IL-10Rα are as follows Figure 6 As shown in the figure, the IL-10 monomer end of fusion proteins R0989 and R0993 has significantly weaker binding activity to IL-10Rα, which may be related to the fact that the insertion position of IL-10 monomer is close to the C-terminal part of the antibody. The binding activity of the remaining fusion proteins to IL-10Rα is relatively close, which is consistent with the positive control molecule R0594. This shows that the IL-10 monomer inserted into the antibody domain can still form the expected spatial structure after recombinant expression and bind to IL-10Rα normally.

[0164] 3.2 The reporter gene method was used to detect the activity of the IL-10 monomer end of the IL-10 monomer fusion protein.

[0165] All fusion protein molecules and the control protein molecule R0579 (IL-10-positive antibody fusion protein control, whose heavy chain amino acid sequence is shown in SEQ ID NO: 23, and whose light chain amino acid sequence is shown in SEQ ID NO: 14, wherein the IL-10 is obtained by removing the first two amino acids of natural IL-10 and without inserting the first connecting peptide) were diluted with 1640 + 10% FBS to an initial concentration of 20 μg / mL, a volume of 450 μl, and a 3-fold gradient dilution (150 μl + 300 μl) to obtain a total of 9 concentrations. Cell preparation: Take out the cultured HEK293-hIL-10-Report gene cells (Cat#GM-C07927, Genomeditech) and observe them under a microscope. Cells with normal cell adhesion, transparent particles and moderate density can be used as effector cells for the experiment; digest the above cells with TE, centrifuge at 300g for 4 minutes after terminating the digestion, remove the supernatant, resuspend in 1% FBS-PBS and wash again; discard the supernatant, and finally resuspend the cells with culture medium. After counting the cells, adjust the cell density to 5×10 5 / mL. According to the above experimental design, the diluted drug was first added to the corresponding well plate at a volume of 50μL / well, and then the above resuspended cells were also added at a volume of 50μL / well. 100μL of culture medium was added to the Medium only wells, and 50μL of culture medium was added to the Cell only wells. The final volume of all wells was 100μL. The 96-well plate continued to be cultured in the incubator for 16h. Thaw the Bright-LumiTM firefly luciferase detection reagent in advance and equilibrate to room temperature. After 16h of cell culture, take out the cell culture plate and equilibrate to room temperature for 10min (not more than 30min). Add 100μL Bright-LumiTM firefly luciferase detection reagent to each well and incubate at room temperature for 5-10min. Use the chemiluminescence mode in the multifunctional microplate reader to detect the signal.

[0166] Test results such as Figure 7 As shown in the figure, the reporter gene results are similar to the binding results. The binding activity of the IL-10 monomer end of the fusion proteins R0989 and R0993 is low, while the activity of the positive control R0579 is much stronger than the fusion protein tested this time. The results of the reporter gene method show that the binding activity of the modified IL-10 monomer is lower than that of the dimer IL-10 molecule.

[0167] Example 4 Effect of fusion protein on MC38 tumor model mice

[0168] The inventors conducted an in vivo experiment on mice based on the detection in Example 3. The specific experimental operation is as follows:

[0169] Cell culture: MC38 tumor cells (source: National Laboratory Cell Resource Sharing Platform, resource number: 3111C0001CCC000523) were cultured in DMEM (supplier: Gibco, catalog number: 11965084) supplemented with 10% FBS (inactivated fetal bovine serum, supplier: Gibco, catalog number: 10091148), 100U / mL penicillin and 100μg / mL streptomycin. The incubator temperature was controlled at 37°C and supplemented with 5% carbon dioxide. Tumor cells were passaged twice a week and digested with trypsin-EDTA.

[0170] Inoculation: MC38 tumor cells in the logarithmic growth phase were collected and centrifuged at 250g for 10 minutes after digestion. After washing twice with ice-cold PBS, the cells were counted and the PBS was adjusted to a cell concentration of 3×10 6 The right abdomen of the mice was shaved in advance and the skin was prepared. Each mouse was inoculated with 0.1 mL of cell suspension, i.e., 3×10 5 A tumor cell.

[0171] Grouping: On days 6-8 after inoculation, tumors of all animals were measured and weighed. To avoid the effect of initial tumor volume on treatment effectiveness, mice were randomly assigned to groups based on tumor volume to ensure that the average tumor volume of all groups was comparable so that the treatment effects were comparable at baseline.

[0172] Observation: After inoculation, the animals were checked for morbidity and mortality every day. After the treatment started, the fusion proteins R0987, R0988, R0989, R0991, R0992, R0993 and R0862 (i.e., Isotype, negative control antibody; its heavy chain amino acid sequence and light chain amino acid sequence are shown in SEQ ID NO: 16, 17), R0428, R0676 (IL-10-Fc fusion protein control, its amino acid sequence is ... As shown in NO:18, wherein the IL-10 removes the first two amino acids of natural IL-10 and does not insert the first connecting peptide)), R0579 protein molecules were intraperitoneally injected into mice at doses of 0.61, 0.61, 0.61, 0.61, 0.61, 0.61, 0.5, 0.5, 0.3, and 0.6 mg / kg, respectively, Q3D×3 (once every 3 days, a total of 3 times), the tumor volume was measured 3 times a week, and the tumor growth of the animals and the effects of treatment on normal behavior, such as mobility, food and water consumption, weight gain / loss, eyes / hair / skin, etc., were examined and recorded.

[0173] Heavy chain sequence of R0862:

[0174] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAAIWYDGSNKYYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLRGVMYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:16).

[0175] The light chain sequence of R0862:

[0176] EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQLRNNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:17).

[0177] R0676 contains two identical peptide chains, and the amino acid sequence of one of the chains:

[0178] DKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLK ESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK NAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO:18).

[0179] Heavy chain sequence of R0579:

[0180] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGAGGGGSGGGGSGGGGSGGGGSG SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKG YLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEK GIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO:23)

[0181] Light chain sequence of R0579:

[0182] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGS

[0183] GTDFLTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLN

[0184] NFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 14).

[0185] Endpoints: The main endpoint of the experiment is whether tumor growth is delayed or whether the mice are cured. TGI (Tumor Growth Inhibition) is calculated based on the tumor volume of each group. Calculation method: Independent sample T test can be used for comparison between two groups. One-Way ANOVA is used for comparison of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. The data were processed using GraphPad Prism, and p<0.05 indicated significant statistical differences. Tumor volume V = 0.5a × b2, a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100, Ti is the average tumor volume of the treatment group on the i-th day, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on the i-th day, and V0 is the average tumor volume of the solvent control group S at the beginning of treatment. TGI is used to represent the effect of the drug on tumor growth inhibition.

[0186] The experimental results are as follows Figure 8 As shown, the tumor growth rate in mice treated with fusion proteins R0987, R0991, and R0993 was reduced and the tumor volume was smaller; compared with monoclonal antibodies or IL-10 fusion proteins, fusion proteins R0987, R0991, and R0993 had better tumor inhibition effects.

[0187] Example 5 Effect of fusion protein on CT26 tumor model mice

[0188] The inventors further verified the fusion protein based on the experimental results obtained in Example 4. The specific experimental operation is as follows:

[0189] Cell culture: CT26 tumor cells (supplier: Chinese Academy of Sciences Cell Bank, catalog number: NA) were cultured in RPMI-1640 (supplier: Gibco, catalog number: 11875085) medium supplemented with 10% FBS (inactivated fetal bovine serum, supplier: Gibco, catalog number: 10091148), 100U / mL penicillin and 100μg / mL streptomycin. The incubator temperature was controlled at 37°C and supplemented with 5% carbon dioxide. Tumor cells were passaged twice a week and digested with trypsin-EDTA.

[0190] Inoculation: Collect cells in the logarithmic growth phase, centrifuge at 250 g for 10 minutes after digestion, wash twice with ice-cold PBS, count, and adjust the cell concentration in PBS to 1×10 6 The right abdomen of the mice was shaved and skin was prepared in advance, and each mouse was inoculated with 0.1 mL of cell suspension, i.e., 1×10 5 A tumor cell.

[0191] Grouping: On days 6-8 after inoculation, tumors of all animals were measured and weighed. To avoid the effect of initial tumor volume on treatment effectiveness, mice were randomly assigned to groups based on tumor volume to ensure that the average tumor volume of all groups was comparable so that the treatment effects were comparable at baseline.

[0192] Observation: The use and care of animals involved in this study protocol were reviewed and approved by the Animal Ethics Committee of Guangdong Feipeng Pharmaceutical Co., Ltd. before the study was conducted. During the study, the care and use of animals were carried out according to the standard operating procedures of the SPF animal house. After inoculation, the animals were checked daily for morbidity and mortality. After the start of treatment, the fusion proteins R0987, R0991, R0993 and R0862, R0579, R0676, R1049 (anti-PD-1 monoclonal antibody control, whose heavy chain amino acid sequence is shown in SEQ ID NO: 19, and the light chain amino acid sequence is shown in SEQ ID NO: 20) protein molecules were intraperitoneally injected into mice at doses of 10, 10, 10, 10, 10, 5, and 7.9 mg / kg, respectively, Q3D×3 (once every 3 days, for a total of 3 times), the tumor volume was measured twice a week, and the tumor growth of the animals and the effects of the treatment on normal behavior, such as mobility, food and water consumption, weight gain / loss, eyes / hair / skin, etc., were examined and recorded.

[0193] R1049 heavy chain sequence:

[0194] QVQLKESGPGLVQPSQTLSLTCTVSGFSLTSYNVHWVRQPPGKGLEWMGGMRYNEDTSYNSALKSRLSISRDTSKNQVFLKMNSLQTDDTGTYYCTRDAVYGGYGGWFAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19).

[0195] R1049 light chain sequence:

[0196] DTVLTQSPALAVSLGQRVTISCKASETVSSSMYSYIHWYQQKPGQQPKLLIYRASNLESGVPARFSGSGSGTDFTLTIDPVEADDVATYFCQQSWNPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:20).

[0197] Endpoints: The main endpoint of the experiment is whether tumor growth is delayed or whether the mice are cured. TGI (Tumor Growth Inhibition) is calculated based on the tumor volume of each group. The calculation method is: the independent sample T test can be used for comparison between two groups. One-Way ANOVA is used for comparison of more than 3 groups. If the F value shows a significant difference, post hoc analysis between multiple groups can be performed. The data are processed using GraphPad Prism, and when p<0.05, it indicates a significant statistical difference. Tumor volume V=0.5a×b2, a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition TGI (%)=[1-(Ti-T0) / (Vi-V0)]×100, Ti is the average tumor volume of the treatment group on the i-th day, T0 is the average tumor volume of the treatment group at the beginning of treatment, Vi is the average tumor volume of the solvent control group on the i-th day, and V0 is the average tumor volume of the solvent control group S at the beginning of treatment. TGI is used to represent the effect of the drug on tumor growth inhibition. According to the specific experiment, the survival curve is also used as one of the criteria for measuring drug efficacy.

[0198] The experimental results are as follows Fig. 9 As shown, after treatment with fusion proteins R0991, R0987, and R0993, the tumor growth rate in mice was significantly reduced, the tumor volume was smaller, and the tumor inhibition effect was better.

[0199] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0200] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that The method comprises an antibody and an IL-10 monomer, wherein the antibody comprises two identical light chains and two identical heavy chains, and an IL-10 monomer is connected to the heavy chain or the light chain of the antibody.

2. The fusion protein according to claim 1, characterized in that Each light chain of the antibody includes a VL region and a CL region, and each heavy chain of the antibody includes a VH region, a CH1 region, a CH2 region, and a CH3 region; each light chain or each heavy chain of the antibody is connected to one IL-10 monomer; Optionally, the N-terminus of the IL-10 monomer is linked to the C-terminus of the light chain of the antibody; or The C-terminus of the IL-10 monomer is connected to the N-terminus of the light chain of the antibody; or The N-terminus of the IL-10 monomer is linked to the C-terminus of the VL region of the antibody light chain, and the C-terminus of the IL-10 monomer is linked to the N-terminus of the CL region of the antibody light chain; or The N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain of the antibody; or The C-terminus of the IL-10 monomer is linked to the N-terminus of the heavy chain of the antibody; or The N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain VH region of the antibody, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH1 region of the antibody; or The N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain CH2 region of the antibody, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH3 region of the antibody.

3. The fusion protein according to claim 1 or 2, characterized in that The IL-10 monomer includes a natural IL-10 monomer; Optionally, the IL-10 monomer comprises a modified natural IL-10 monomer, wherein the modification comprises inserting a first connecting peptide between amino acids 116 and 117, wherein the first connecting peptide is a flexible connecting peptide; Optionally, the first connecting peptide has the amino acid sequence shown in SEQ ID NO: 21; Optionally, the IL-10 monomer has the amino acid sequence shown in SEQ ID NO:

2.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The IL-10 monomer is connected to the heavy chain or light chain of the antibody via a second connecting peptide and / or a third connecting peptide; Optionally, the second connecting peptide has the amino acid sequence shown in SEQ ID NO:12, and the third connecting peptide has the amino acid sequence shown in SEQ ID NO:

22.

5. The fusion protein according to claim 4, characterized in that The IL-10 monomer is connected to the heavy chain end or the light chain end of the antibody via the second connecting peptide; optionally, The N-terminus of the second connecting peptide is connected to the C-terminus of the light chain of the antibody, and the C-terminus of the second connecting peptide is connected to the N-terminus of the IL-10 monomer; or The N-terminus of the second connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the light chain of the antibody; or The N-terminus of the second connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the second connecting peptide is connected to the N-terminus of the heavy chain of the antibody; or The IL-10 monomer is connected to the heavy chain end or the light chain end of the antibody through the third connecting peptide; the N-terminus of the third connecting peptide is connected to the C-terminus of the light chain of the antibody, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or The N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the light chain of the antibody; or The N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain of the antibody, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or The N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain of the antibody; or The N-terminus of the third connecting peptide is connected to the C-terminus of the light chain VL region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or The N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the light chain CL region; or The N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain VH region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or The N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain CH1 region; or The N-terminus of the third connecting peptide is connected to the C-terminus of the heavy chain CH2 region, and the C-terminus of the third connecting peptide is connected to the N-terminus of the IL-10 monomer; or The N-terminus of the third connecting peptide is connected to the C-terminus of the IL-10 monomer, and the C-terminus of the third connecting peptide is connected to the N-terminus of the heavy chain CH3 region; or The N-terminus of the IL-10 monomer is connected to the C-terminus of the VL region of the light chain of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the CL region of the light chain of the antibody through the second connecting peptide; or The N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain VH region of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH1 region of the antibody through the second connecting peptide; or The N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain CH2 region of the antibody through the second connecting peptide, and the C-terminus of the IL-10 monomer is connected to the N-terminus of the heavy chain CH3 region of the antibody through the second connecting peptide.

6. The fusion protein according to any one of claims 1 to 5, characterized in that The antibody is selected from immune cell antibodies and tumor cell antibodies; Optionally, the antibody is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Her2 antibody, an anti-CCR8 antibody, an anti-VEGFR2 antibody, an anti-claudin18.2 antibody, an anti-CD39 antibody, an anti-SMA4D antibody, an anti-GUCY2C antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, an anti-CD47 antibody or an anti-TNFα antibody; Optionally, the antibody is an anti-PD-1 antibody.

7. The fusion protein according to any one of claims 1 to 6, characterized in that The fusion protein has the amino acid sequences described in SEQ ID NO: 3 and SEQ ID NO: 9; or The fusion protein has the amino acid sequences described in SEQ ID NO: 3 and SEQ ID NO: 5; or The fusion protein has the amino acid sequences described in SEQ ID NO:4 and SEQ ID NO:6; or The fusion protein has the amino acid sequences described in SEQ ID NO:7 and SEQ ID NO:6; or The fusion protein has the amino acid sequences described in SEQ ID NO: 3 and SEQ ID NO: 8; or The fusion protein has the amino acid sequences described in SEQ ID NO: 11 and SEQ ID NO: 6; or The fusion protein has the amino acid sequences described in SEQ ID NO:10 and SEQ ID NO:

6.

8. A biomaterial, characterized in that The biological material comprises the following (i), (ii) or (iii): (i) a nucleic acid encoding the fusion protein according to any one of claims 1 to 7; (ii) an expression vector comprising the nucleic acid described in (i); (iii) A recombinant cell, characterized in that the recombinant cell carries the nucleic acid described in (i), the expression vector described in (ii), or expresses the fusion protein described in any one of claims 1 to 7.

9. A pharmaceutical composition, characterized in that Comprising the fusion protein according to any one of claims 1 to 7 and the biomaterial according to claim 8.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is used for treating or preventing tumors or inflammatory diseases.

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