CN114316064A

CN114316064A - Fusion protein and application thereof

Info

Publication number: CN114316064A
Application number: CN202111158800.2A
Authority: CN
Inventors: 霍永庭; 韩喆; 张丽丽; 张喆; 罗甜; 路力生
Original assignee: Guangdong Fapon Biopharma Inc
Current assignee: Guangdong Fapon Biopharma Inc
Priority date: 2020-10-10
Filing date: 2021-09-30
Publication date: 2022-04-12

Abstract

The invention provides a fusion protein. The fusion protein comprises: a VEGF antagonist antibody comprising a Fab region and an Fc region, and IL-10. The inventor finds that the killing effect of the fusion protein on the tumor is obviously better than the combination of a single VEGF antagonistic antibody and a single empty target antibody-IL-10 fusion protein, and the synergy of the VEGF antagonistic antibody and the IL-10 fusion protein is obvious.

Description

Fusion protein and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to fusion protein and application thereof, and more particularly relates to fusion protein of an anti-VEGFR (or VEGF) antibody and interleukin-10 (IL-10), and nucleic acid, a construct, a recombinant cell, a pharmaceutical composition and pharmaceutical application thereof for encoding the fusion protein.

Background

Angiogenesis refers to the formation of capillaries from blood vessels that are pre-existing in embryonic and adult organisms, and is known to be an important factor in tumor growth, survival and metastasis. VEGF is an isoform, a dimeric glycoprotein composed of two 23kD subunits, and it is a strong vascular permeability inducer, a stimulator of endothelial cell migration and proliferation, and an important survival factor for newly formed blood vessels. VEGF receptors (VEGFRs), which are typically class III receptor type tyrosine kinases, VEGFR-1(flt-1), VEGFR-2(KDR/flk-1) and VEGFR-3(flt-4), block the interaction between VEGF and its receptor, and inhibit angiogenesis and tumor growth.

Human interleukin-10 (hIL-10) cytokine, an alpha-helical factor, is expressed as a non-covalently linked homodimer of about 37kDa and exerts its biological activity. Mosmannand, first reported in 1989, is a pleiotropic cytokine with a bi-directional regulation of the immune system that regulates a variety of immune responses through actions on T cells, B cells, macrophages and antigen presenting cells. In one aspect, IL-10 can inhibit an immune response primarily associated with inflammation by inhibiting the expression of IL-1 α, IL-1 β, IL-6, IL-8, TNF- α, GM-CSF and G-CSF in activated monocytes and activated macrophages; on the other hand, John B.Mumm et al found that IL-10 could induce and stimulate tumor-infiltrating CD8+ T to secrete cytotoxic cytokine gamma interferon (IFN-gamma), and further mediate the production of cytotoxic substance granzyme B (Granzyme B), perforin (perfolin) and the like, thereby inhibiting and killing tumor cells and exhibiting corresponding immunostimulatory effect.

Based on available clinical data, hIL-10 exhibits a very short plasma half-life-a half-life of only 2.5 hours in the systemic compartment-due to its small size, which is rapidly cleared by the kidneys. To improve circulation time, exposure, efficacy and reduce renal uptake, many studies have reported pegylation of IL-10. The dose-dependent antitumor activity of pegylated IL-10(PEG-IL-10, AM0010) was also reported by ARMO biosciens, inc. But PEGylated hIL-10 is not targeted, and the longer half-life also aggravates the adverse events of the molecule.

Antibody-cytokine fusion proteins (also called immunocytokines) are of great interest because of their targeting properties and their ability to reduce toxic side effects, and currently, few enterprises are in development of antibody drugs (including fusion proteins) targeting IL-10 globally, and most of them are used for treating autoimmune diseases, and only Y-Biologics and chinese corning jierie, which are known as development products for cancer treatment, are in the development (discovery) stage.

The reason for this is that it is difficult to find an antibody target capable of effectively fusing with interleukin-10, and the effect is not good after many target antibodies and interleukin-10 are made into fusion protein.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

the inventors of the present application inventively fused IL-10 with antibodies to VEGFRs (or VEGF) to obtain fusion proteins. The inventors surprisingly found that the obtained fusion protein achieved synergistic interaction.

In a first aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, the fusion protein comprises: a VEGF antagonist antibody comprising a Fab region and an Fc region, and IL-10. It should be noted that the Fab region and the Fc region of the antibody described herein refer to two identical Fab fragments and one Fc fragment obtained assuming that the antibody is hydrolyzed by papain. A VEGF antagonist antibody as described herein refers to an antibody that antagonizes or inhibits the effects of VEGF associated with VEGFRs. The IL-10 described herein includes naturally occurring IL-10 or modified IL-10. The inventor finds that the killing effect of the fusion protein on the tumor is obviously better than that of a single VEGF antagonistic antibody and a single empty target antibody-IL-10 fusion protein, and the synergistic effect of the VEGF antagonistic antibody and the IL-10 fusion protein is obvious.

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

according to an embodiment of the invention, the VEGF antagonist antibody comprises at least one selected from the group consisting of anti-VEGF antibodies and anti-VEGFRs antibodies.

According to an embodiment of the invention, the VEGF comprises at least one selected from the group consisting of VEGF-A, VEGF-B, PlGF, VEGF-A, VEGF-B, VEGF-E, PlGF-2, VEGF-A, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGF-A, VEGF-C, PlGF-2, VEGF-C, VEGF-D.

According to embodiments of the invention, the VEGFRs include at least one selected from the group consisting of VEGFR1, NRP-1, VEGFR2, NRP-2, VEGFR 3.

According to embodiments of the invention, the anti-VEGFRs antibody is an anti-VEGFR2 antibody. The inventor finds that after the anti-VEGFR2 antibody and IL-10 form a fusion protein, the synergistic effect of the anti-VEGFR2 antibody and IL-10 is more remarkable.

According to an embodiment of the invention, the anti-VEGFR antibody comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity thereto: heavy chain variable region CDR sequences: 1-3 of SEQ ID NO, and a light chain variable region CDR sequence: 4-6 of SEQ IN NO.

SYSMN(SEQ ID NO:1)。

SISSSSSYIYYADSVKG(SEQ ID NO:2)。

VTDAFDI(SEQ ID NO:3)。

RASQGIDNWLG(SEQ ID NO:4)。

DASNLDT(SEQ ID NO:5)。

QQAKAFPPT(SEQ ID NO:6)。

According to an embodiment of the invention, the anti-VEGFRs antibody comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in

SEQ ID NOs

1, 2 and 3, respectively, or amino acid sequences at least 95% identical to

SEQ ID NOs

1, 2 and 3, and light chain variable region CDR1, CDR2, CDR3 sequences as set forth in

SEQ ID NOs

4, 5 and 6, respectively, or amino acid sequences at least 95% identical to

SEQ ID NOs

4, 5 and 6.

According to embodiments of the invention, the anti-VEGFRs antibody has the amino acid sequence as set forth in SEQ ID NO: 7, or a heavy chain variable region of the amino acid sequence shown in seq id no.

EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTVSS(SEQ ID NO:7)。

According to embodiments of the invention, the anti-VEGFRs antibody has the amino acid sequence as set forth in SEQ ID NO: 8 in a light chain variable region.

DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSG SGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKR(SEQ ID NO:8)。

According to embodiments of the invention, the anti-VEGFRs antibody comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

According to embodiments of the invention, the light chain constant region and the heavy chain constant region of the anti-VEGFRs antibody are both from a human IgG antibody or a mutant thereof. Further, the immunogenicity of the antibody can be effectively reduced.

According to embodiments of the invention, the light chain constant region and the heavy chain constant region of the anti-VEGFRs antibody are both from human IgG 1.

According to an embodiment of the invention, the Fc region has the a327Q, G237A, and L235A mutations. The inventor finds that the Fc region has A327Q, G237A and L235A mutations, so that the binding of Fc to Fc receptors (FcRs) can be greatly reduced, the ADCC effect is reduced, and the toxic and side effects of the medicine are further reduced.

According to an embodiment of the invention, said Fc region further has the K447A mutation. Thereby preventing the enzymolysis of the C-terminal connexin and enhancing the stability of the fusion protein.

According to an embodiment of the present invention, the Fc region has the amino acid sequence shown in SEQ ID NO 9.

EPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKQLPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO:9)。

The first amino acid in the amino acid sequence shown in SEQ ID NO. 9 is position 216 in Eu number (Eu numbering).

According to an embodiment of the present invention, the anti-VEGFRs antibody has a heavy chain having the amino acid sequence shown in SEQ ID No. 10 and a light chain having the amino acid sequence shown in SEQ ID No. 11.

EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKQLPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO:10)。

DIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLIYDASNLDTGVPSRFSGSG SGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC(SEQ ID NO:11)。

According to an embodiment of the present invention, the fusion protein further comprises a linker peptide, wherein the N-terminus of the linker peptide is linked to the C-terminus of the VEGF antagonist antibody, and the C-terminus of the linker peptide is linked to the N-terminus of the IL-10. The connecting peptide connects two proteins with different functions, keeps a certain distance, and can exert respective biological functions without mutual influence.

According to an embodiment of the present invention, the amino acid sequence of the linker peptide has the amino acid sequence shown in SEQ ID NO. 12.

GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:12)。

According to a specific embodiment of the invention, the IL-10 has the amino acid sequence shown in SEQ ID NO 13.

SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQAL SEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQE KGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:13)。

In a second aspect of the invention, a fusion protein is provided. According to an embodiment of the invention, the fusion protein comprises: a targeting antibody comprising a Fab region and an Fc region having the a327Q, G237A and L235A mutations, and IL-10. The term "targeting antibody" as used herein refers to an antibody capable of specifically targeting and binding to an antigen. According to the fusion protein disclosed by the embodiment of the invention, the aggregation of IL-10 in a targeting region is realized under the mediation of a targeting antibody, the drug effect is increased, and the half-life of IL-10 is effectively prolonged, more importantly, the fusion protein disclosed by the embodiment of the invention has weak binding force with an Fc receptor (FcR), the ADCC effect is low, and the toxic and side effects of a drug are remarkably reduced.

according to embodiments of the invention, the targeting antibody is an antibody targeting VEGF or VEGFRs. The inventors surprisingly found that after an antibody targeting VEGF or VEGFRs forms a fusion protein with IL-10, the two antibodies show significant synergy.

According to an embodiment of the invention, the VEGFR includes at least one selected from the group consisting of VEGFR1, NRP-1, VEGFR2, NRP-2, VEGFR 3.

According to an embodiment of the invention, the targeting antibody is a VEGFR2 targeting antibody. The inventors found that after the VEGFR 2-targeting antibody and IL-10 form a fusion protein, the synergistic effect of the two is more remarkable.

According to embodiments of the invention, the targeting antibody specifically binds to the extracellular region of VEGFR.

According to an embodiment of the invention, the targeting antibody comprises a CDR sequence or an amino acid sequence with at least 95% identity thereto selected from at least one of the following: heavy chain variable region CDR sequences: 1-3 of SEQ ID NO, and a light chain variable region CDR sequence: 4-6 of SEQ IN NO.

According to an embodiment of the invention, the targeting antibody comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in

SEQ ID NOs

1, 2 and 3, respectively, or amino acid sequences at least 95% identical to

SEQ ID NOs

4, 5 and 6, respectively, or amino acid sequences at least 95% identical to

SEQ ID NOs

4, 5 and 6.

According to an embodiment of the invention, the targeting antibody has the amino acid sequence as shown in SEQ ID NO: 7, or a heavy chain variable region of the amino acid sequence shown in seq id no.

According to an embodiment of the invention, the targeting antibody has the amino acid sequence as shown in SEQ ID NO: 8 in a light chain variable region.

According to an embodiment of the invention, the targeting antibody comprises a heavy chain constant region comprising the Fc region and a light chain constant region, at least a portion of which is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

According to an embodiment of the invention, the light chain constant region and the heavy chain constant region of the targeting antibody are both from a human IgG antibody. Further, the immunogenicity of the antibody can be effectively reduced.

According to an embodiment of the invention, the light chain constant region and the heavy chain constant region of the targeting antibody are both from human IgG 1.

According to an embodiment of the present invention, the targeting antibody has a heavy chain having an amino acid sequence shown in SEQ ID NO. 10 and a light chain having an amino acid sequence shown in SEQ ID NO. 11.

According to an embodiment of the present invention, the fusion protein further comprises a linker peptide, wherein the N-terminus of the linker peptide is linked to the C-terminus of the targeting antibody, and the C-terminus of the linker peptide is linked to the N-terminus of the IL-10. The connecting peptide connects two proteins with different functions, keeps a certain distance, and can exert respective biological functions without mutual influence.

GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:12)。

In a third aspect of the invention, a nucleic acid molecule is presented. According to an embodiment of the invention, the nucleic acid encodes a fusion protein as described above. After the nucleic acid of the embodiment of the invention is introduced into the receptor cell, the fusion protein is expressed, and the specific killing of the tumor cell is realized.

According to an embodiment of the present invention, the above-mentioned nucleic acid molecule may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the nucleic acid molecule has the nucleotide sequence shown as

SEQ ID NO

14 or 15.

GAGGTGCAGCTGGTGCAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGCGCCTGA GCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGC AAGGGCCTGGAGTGGGTGAGCAGCATCAGCAGCAGCAGCAGCTACATCTACTACGCCGACAGCGT GAAGGGCCGCTTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCC TGCGCGCCGAGGACACCGCCGTGTACTACTGCGCCCGCGTGACCGACGCCTTCGACATCTGGGGC CAGGGCACCATGGTGACCGTGAGCAGCGCCTCTACCAAGGGACCCTCTGTGTTTCCTCTGGCTCCC TCCAGCAAGTCTACCTCTGGTGGAACAGCTGCCCTGGGCTGCCTGGTCAAGGATTACTTTCCTGAG CCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTG CAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGA CCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAG AGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGGCCGGCGCCCCCAGCGT GTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGT GGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCG TGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAA GCAGCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAG GTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGT GAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACT ACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTG GACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACA ACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCGCCGGCGGAGGCGGCAGCGGTGGCGG TGGCAGCGGAGGCGGCGGCAGCGGAGGTGGAGGCAGCGGCTCCCCCGGCCAGGGCACCCAGAGC GAGAACTCTTGTACCCACTTCCCCGGCAACCTGCCCAATATGCTGCGGGATCTGAGAGACGCCTTC AGCAGAGTGAAGACCTTCTTTCAGATGAAGGACCAGCTGGATAACCTGCTGCTGAAGGAGAGCCT GCTGGAGGATTTCAAGGGCTACCTGGGCTGTCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGA GGAGGTAATGCCACAGGCCGAGAATCAGGACCCAGACATCAAGGCCCACGTGAACTCTCTGGGCG AGAATCTGAAGACCCTGAGACTGAGGCTGAGACGCTGTCACAGATTCCTGCCATGCGAGAACAAG AGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAATAAGCTGCAGGAGAAGGGCATCTACAAGG CCATGTCTGAGTTCGACATCTTTATCAACTACATCGAGGCCTACATGACCATGAAGATCCGGAAC (SEQ ID NO:14)。

GACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCATCGGCGACCGCGTGACCAT CACCTGCCGCGCCAGCCAGGGCATCGACAACTGGCTGGGCTGGTACCAGCAGAAGCCCGGCAAGG CCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGACACCGGCGTGCCCAGCCGCTTCAGCGGC AGCGGCAGCGGCACCTACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACTTCGCCGTGTA CTTCTGCCAGCAGGCCAAGGCCTTCCCCCCCACCTTCGGCGGCGGCACCAAGGTGGACATCAAGA GAACTGTGGCCGCTCCATCCGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAGAGTGGCACCG CCTCTGTGGTCTGTCTGCTGAACAATTTCTACCCCCGTGAGGCAAAGGTGCAGTGGAAAGTCGATA ACGCCCTGCAGTCCGGAAATAGCCAGGAGTCTGTGACAGAACAGGACAGTAAGGATTCAACTTATT CTCTGTCTAGTACCCTGACACTGTCTAAAGCTGACTACGAGAAGCACAAAGTGTATGCATGCGAAG TCACCCATCAGGGGCTGTCATCCCCTGTGACAAAGTCCTTTAATCGCGGTGAATGT(SEQ ID NO:15)。

In a fourth aspect of the invention, the invention features a construct. According to an embodiment of the invention, the construct carries a nucleic acid as described above. After the construct according to the embodiment of the invention is introduced into the recipient cell, the carried nucleic acid is integrated or not integrated with the genome of the recipient cell, and the fusion protein is expressed, so that the specific killing of the tumor cell is realized.

According to an embodiment of the present invention, the above-mentioned construct may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the vector of the construct is PCDNA3.4A.

According to an embodiment of the present invention, further comprising a promoter operably linked to the nucleic acid.

In a fifth aspect of the invention, a recombinant cell is provided. According to an embodiment of the invention, said recombinant cell carries a nucleic acid as described above or a construct as described above. The recombinant cell according to the embodiment of the invention expresses the fusion protein, and realizes specific killing on tumor cells.

In a sixth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises the fusion protein as described above, the nucleic acid as described above, the construct as described above or the recombinant cell as described above. The pharmaceutical composition provided by the embodiment of the invention has a more significant killing effect on tumors.

According to an embodiment of the present invention, the above pharmaceutical composition may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In a seventh aspect, the present invention provides the use of a fusion protein as defined above, a nucleic acid as defined above, a construct as defined above, a recombinant cell as defined above or a pharmaceutical composition as defined above for the manufacture of a medicament for the treatment or prevention of a vascular proliferative disease, an autoimmune disease or a tumour. The fusion protein or the fusion protein expressed by the nucleic acid, the construction body and the recombinant cell can specifically target VEGF or VEGFRs, and the combination of VEGF and VEGFRs has the effect of promoting vascular proliferation, so that the medicine prepared by the fusion protein according to the embodiment of the invention can be used for obviously inhibiting vascular proliferation and has the effect of treating or preventing vascular proliferative diseases, meanwhile, IL10 in the fusion protein according to the embodiment of the invention can specifically bind to an IL10 receptor, inhibit the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, GM-CSF and G-CSF in activated monocytes and activated macrophages to inhibit immune responses related to inflammation and induce and stimulate the infiltration of tumor CD8+ T to secrete cytotoxic cell factor gamma interferon (IFN-gamma), further mediates the generation of cell toxic substance granzyme B (granzyme B), perforin (perfolin) and the like, thereby inhibiting and killing tumor cells, therefore, the medicine prepared by the fusion protein according to the embodiment of the invention can be used for inhibiting transitional immunity and killing tumor cells, and has the treatment or prevention effect on autoimmune diseases or tumors.

The fusion protein provided by the embodiment of the invention has the following advantages:

1. compared with natural IL10, the compound has longer half-life period in vivo, so the antitumor effect is better;

2. compared with the IL10 fused with Fc fragment, the micro-tumor-targeting fusion protein can realize the accurate delivery of the tumor microenvironment, so the dosage is smaller, and the toxic and side effects of the drug are smaller under the condition of better curative effect;

3. compared with the Fc fragment fused IL10, the Fc fragment fused IL10 has better druggability and more stable expression;

4. point mutation is carried out on the Fc segment, so that the binding of the Fc segment with an Fc receptor (FcR) is greatly reduced, the ADCC effect is reduced, and the toxic and side effects of the medicine are further reduced;

5. has stronger tumor inhibiting effect than the anti-VEGF antibody or the anti-VEGFRs antibody under the same dosage;

6. compared with an anti-VEGF antibody or an anti-VEGFRs antibody and IL10 alone, the anti-VEGF antibody or the anti-VEGFRs antibody has stronger tumor inhibiting effect under the same dosage.

Drawings

FIG. 1 is a drawing of the various molecular structures and codes involved in this patent;

FIG. 2a is a map of an PCDNA3.4A plasmid according to an embodiment of the present invention;

FIG. 2b is a map of the plasmid PCDNA3.4A-R0467VCHE according to an embodiment of the present invention;

FIG. 2c is an PCDNA3.4A-R0467VCLE plasmid map according to an embodiment of the present invention;

FIG. 2d is a map of the PCDNA3.4A-R0468VCHE plasmid according to an embodiment of the present invention;

FIG. 2e is an PCDNA3.4A-R0523VCHE plasmid map according to an embodiment of the present invention;

FIG. 2f is an PCDNA3.4A-R0523VCLE plasmid map according to an embodiment of the present invention;

FIG. 2g is a map of PCDNA3.4A-R0568 plasmid according to an embodiment of the present invention;

FIG. 2h is a map of PCDNA3.4A-R0595 plasmid according to an embodiment of the present invention;

FIG. 3 is a SEC detection map after R0467 purification according to an embodiment of the invention;

FIG. 4 is a graph showing the results of an experiment for detecting the binding of each molecule to IL-10R by ELISA according to an embodiment of the present invention;

FIG. 5 is a graph showing the results of an experiment for detecting the binding of each molecule to CHO-IL10R by a flow method according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of an experiment for detecting the activity of IL10 terminal of each molecule according to the reporter gene method of the present invention;

FIG. 7 is the biological activity (non-immobilized) of U937 secreting TNF- α according to an embodiment of the present invention;

FIGS. 8a-8c are graphs showing the biological activity of inhibiting the secretion of TNF-. alpha.by U937 after immobilization of R0467 according to an embodiment of the present invention, wherein IC50-1 in FIG. 8a is the result of the first experiment in example 8; IC50-2 in FIG. 8b is the result of the second experiment in example 8; IC50-3 in FIG. 8c is the result of the third experiment in example 8;

FIG. 9 shows that ELISA according to the present invention detects the binding activity of Cyramza end of each molecule to VEGFR 2;

FIG. 10 shows that the activity of each Cyramza end-blocked VEGF165 and VEGFR2 by ELISA according to the present invention;

FIG. 11 is an evaluation of the antitumor activity of R0467 in the B16-F1 model (first experiment) according to an embodiment of the present invention;

FIG. 12 is an evaluation of the antitumor activity of R0467 in the B16-F1 model (second experiment) according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Antibodies

Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprises two light chains with lighter molecular weight and two heavy chains with heavier molecular weight, wherein the heavy chains (H chains) and the light chains (L chains) are connected by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies widely and is called variable region (V region), and the carboxy-terminal (C-terminal) is relatively stable and varies little and is called constant region (C region). The V regions of the L chain and the H chain are respectively called VL

And VH. In one embodiment, the antibody is of the IgG class, in one embodiment the IgG class antibody is an IgG1 antibody.

Certain regions in the variable region, which have a higher degree of variation in amino acid composition and arrangement order, are called Hypervariable regions (HVRs), which are the sites where antigens and antibodies bind and are therefore also called complementarity-determining regions (CDRs). The heavy chain variable region and the light chain variable region both have three CDR regions.

The C regions of the heavy and light chains are referred to as CH and CL, respectively. The CL lengths of different classes (κ or λ) of igs are essentially identical, but the CH lengths of different classes of igs are different, e.g., IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CHl, CH2, CH3, and CH 4.

The hinge region (hinge region) is located between CH1 and CH2, is proline-rich, and is easily extended and bent, thereby changing the distance between antigen binding sites, facilitating the binding of antibodies to epitopes located at different positions. The hinge region is susceptible to hydrolysis by papain, pepsin, etc., resulting in different hydrolyzed fragments.

The papain hydrolyzes Ig at a site near the N-terminus of two heavy chains disulfide-linked in the hinge region, cleaving Ig into two identical Fab fragments and an Fc fragment.

The Fab fragment, the antigen binding fragment (Fab), consists of an intact light chain and the VH and CHl domains of the heavy chain. The Fab fragments in this application target VEGF or VEGF receptors (VEGFRs).

The VEGF (vascular endothelial growth factor) family includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, PlGF (placental growth factor) and VEGF-F. Vascular endothelial growth factors exert their respective biological functions by being associated with their receptors. These receptors include the transmembrane tyrosine kinase receptors VEGFR1, VEGFR2, VEGFR3, and neuropilin receptors (NRP-1 and NRP-2). VEGF family members play an important role in inducing the formation of blood and lymphatic vessels.

Some examples of VEGF receptors include protein tyrosine kinase receptors such as flt-1(VEGFR-1), KDR and flk-1(VEGFR-2) mentioned in the literature. Unless otherwise stated or clearly stated otherwise, the specification will follow the conventional literature nomenclature for VEGF receptors.

VEGF receptors (VEGFRs)	VEGF (VEGF ligand)
		VEGFR1	VEGF-A、VEGF-B、PlGF
NRP-1	VEGF-A、VEGF-B、VEGF-E、PlGF-2
		VEGFR2	VEGF-A、VEGF-C、VEGF-D、VEGF-E、VEGF-F
NRP-2	VEGF-A、VEGF-C、PlGF-2
		VEGFR3	VEGF-C、VEGF-D、

Blocking the interaction between VEGF and its receptor can inhibit angiogenesis and tumor growth.

In some embodiments, the present invention provides a fusion protein comprising an anti-VEGFR2 antibody having the amino acid sequence of SEQ ID NO: 1-3 and SEQ IN NO 4-6.

The Fc fragment, i.e., the crystallizable (Fc), consists of the CH2 and CH3 domains of Ig. The Fc region has no antigen binding activity and is the site of interaction of Ig with effector molecules or cells. The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC). These respective functions are mostly associated with Fc binding to Fc receptors (Fc γ receptors) including Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32) and Fc α RI (CD 89).

Antibodies comprising modifications that reduce Fc receptor binding typically have reduced effector function, particularly reduced ADCC, as compared to the corresponding unmodified antibody. Thus, in one embodiment, the modification that reduces the binding affinity of an IgG class antibody to an Fc receptor reduces the effector function of the IgG class antibody. In a specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

Without being limiting to the present application, any Fc variant with reduced affinity for Fc γ receptors reported in the prior art may be applied in the preparation of fusion proteins, for example, the entire content of patent application PCT/CN2020/118409 is incorporated herein, the Fc variant being selected from E223P, P238A, D265A; N297A, L235A, N434A; or a327Q, G237A, L235A; common Fc variants also contain a deletion at position K447, or in order to avoid enzymatic cleavage of other proteins at the C-terminus of the Fc leading to mutation at K447A, the deletion or mutation at position 447 of the Fc region does not affect the affinity of the Fc variant for Fc γ receptors and/or FcRn. Note that the Fc region numbers presented herein are based on Eu numbers (Eu numbering).

"native IL-10" (also referred to as "wild-type IL-10") means a naturally occurring IL-10, as opposed to a "modified IL-10" which has been modified from the naturally occurring IL-10, for example in order to alter one or more of its properties, such as stability. The modified IL-10 molecule may, for example, comprise a modification in the amino acid sequence, such as an amino acid substitution, deletion or insertion. One particular modified IL-10 molecule with increased stability in monomeric form has been described (Josephson et al, J Biol Chem275,13552-13557 (2000)).

The term "linker peptide" is a peptide comprising one or more amino acids, typically about 2-20 amino acids. Linking peptides are known in the art or described herein. Suitable, non-immunogenic linker peptides include, for example, (G)₄S)_n、(SG₄)_nOr G₄(SG₄)_nA peptide linker. "n" is generally a number between 1 and 10, typically between 2 and 4.

Nucleic acid molecules, constructs, recombinant cells

In the process of preparing or obtaining the fusion protein described herein, the nucleic acid molecules expressing these fusion proteins can be used to link with different vectors and then expressed in different cells to obtain the corresponding fusion protein.

To this end, the invention also provides an isolated nucleic acid molecule encoding the above-described fusion protein.

In some embodiments, the isolated nucleic acid molecule has a nucleotide sequence as set forth in

SEQ ID NO

14 or 15. Wherein, the nucleotide shown in SEQ ID NO. 14 encodes the part of the anti-VEGFRs antibody heavy chain-connecting peptide-IL-10 in the fusion protein, and the nucleotide shown in SEQ ID NO. 15 encodes the part of the anti-VEGFR antibody light chain in the fusion protein. The structural diagram of the fusion protein can refer to the structure E in FIG. 1.

In some embodiments, the isolated nucleic acid molecule is at least 90% homologous, preferably 95% homologous, and more preferably 98% or 99% homologous to the nucleotide sequence set forth in SEQ ID NO. 14 or 15 above.

The present invention also provides an expression vector comprising the isolated nucleic acid molecule described above. When the isolated polynucleotide is ligated to a vector, the polynucleotide may be ligated to control elements on the vector directly or indirectly, so long as the control elements are capable of controlling the translation, expression, etc. of the polynucleotide. Of course, these control elements may be derived directly from the vector itself, or may be exogenous, i.e., not derived from the vector itself. Of course, the polynucleotide may be operably linked to a control element. "operably linked" herein refers to the attachment of a foreign gene to a vector such that control elements within the vector, such as transcriptional and translational control sequences and the like, are capable of performing their intended function of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the fusion proteins may be inserted into different vectors, and usually into the same vector. Commonly used vectors may be, for example, plasmids, phages and the like. For example a Plasmid-X.

The invention also provides a recombinant cell which contains the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, and then the recombinant cells are used to express the fusion protein provided by the invention. The corresponding fusion protein can be obtained by culturing the recombinant cell.

Pharmaceutical composition and pharmaceutical use

The invention also provides a pharmaceutical composition, which comprises the fusion protein and a pharmaceutically acceptable carrier.

When a disease is treated using the fusion protein provided by the present invention, the fusion protein provided by the present invention may be provided to a subject. To this end, the present invention provides a method for treating the above-mentioned diseases, comprising administering to a subject in need thereof a fusion protein provided by the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The structures and symbols of the molecules involved in the following examples are shown in FIG. 1. Wherein the structure A in FIG. 1 represents the IL-10 protein, numbered R0568; the structure B represents an Fc-IL10 fusion protein, No. R0595; the C structure represents an ISO-IL10 fusion protein, No. R0523, wherein ISO represents the antibody moiety of equivalent E structure without targeting, i.e. an empty targeting antibody; structure D represents an anti-VEGFR2 antibody, No. R0468; the structure E shows the fusion protein obtained by fusing IL-10 to the anti-VEGFR2 antibody, numbered R0467. As an illustration, in an embodiment the anti-VEGFR2 antibody is ramucirumab (Cyramza).

Example 1 anti-VEGFR 2-IgG1Fc-wthIL10 antibody fusion protein molecule construction

The purpose is as follows: the Fc segment of human IgG1 antibody is site-directed mutated according to EU numbering mode to construct PCDNA3.4A-R0467, including PCDNA3.4A-R0467VCHE and PCDNA3.4A-R0467VCLE two plasmids, and the corresponding molecular structure of the target fragment is as follows: R0467H: IgG universal signal peptide-Cyramza VH-hIgG1 (containing K447A, a327Q, G237A, L235A mutations) -GGGGSGGGGSGGGGSGGGGSG-hIL 10; R0467L: IgG general signal peptide-Cyramza VH-hIgKC; simultaneously constructing isotype control PCDNA3.4A-R0468 comprising PCDNA3.4A-R0468VCHE, PCDNA3.4A-R0468VCLE (same as PCDNA3.4A-R0467 VCLE); PCDNA3.4A-R0523, including PCDNA3.4A-R0523VCHE and PCDNA3.4A-R0523 VCLE; PCDNA3.4A-R0568 and PCDNA3.4A-R0595, the molecular structure is as follows: R0468H: IgG universal signal peptide-Cyramza VH-hIgG1 (containing K447A, a327Q, G237A, L235A mutations); R0523H: IgG universal signal peptide-Isotype VH-hIgG1 (containing K447A, a327Q, G237A, L235A mutations) -GGGGSGGGGSGGGGSGGGGSG-hIL 10; R0523L: IgG general signal peptide-Isotype VH-hIgKC; r0568: IgG generic signal peptide-6 × HIS-hILl 0; r0595: . The polypeptides expressed by isotype controls PCDNA3.4A-R0468, PCDNA3.4A-R0523, PCDNA3.4A-R0568 and PCDNA3.4A-R0595 are R0468, R0523, R0568 and R0595, respectively. The plasmid map constructed above is shown in FIG. 2, and the nucleotide sequence of the nucleic acid encoding each fusion protein and the amino acid sequence of each fusion protein are as follows.

R0467VCHE gene sequence (1938bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTGAGGTGC AGCTGGTGCAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGC CAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGG AGTGGGTGAGCAGCATCAGCAGCAGCAGCAGCTACATCTACTACGCCGACAGCGTGAAGGGCCGC TTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGA GGACACCGCCGTGTACTACTGCGCCCGCGTGACCGACGCCTTCGACATCTGGGGCCAGGGCACCAT GGTGACCGTGAGCAGCGCCTCTACCAAGGGACCCTCTGTGTTTCCTCTGGCTCCCTCCAGCAAGTC TACCTCTGGTGGAACAGCTGCCCTGGGCTGCCTGGTCAAGGATTACTTTCCTGAGCCTGTGACCGT GTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGG CCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGC AATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACA AGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGGCCGGCGCCCCCAGCGTGTTCCTGTTCC CCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGAC GTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACG CCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTG CTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGCAGCTGCCCG CCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTG CCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTA CCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC CCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCG CTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGAGCCTGAGCCTGAGCCCCGGCGCCGGCGGAGGCGGCAGCGGTGGCGGTGGCAGCGGAGG CGGCGGCAGCGGAGGTGGAGGCAGCGGCTCCCCCGGCCAGGGCACCCAGAGCGAGAACTCTTGT ACCCACTTCCCCGGCAACCTGCCCAATATGCTGCGGGATCTGAGAGACGCCTTCAGCAGAGTGAAG ACCTTCTTTCAGATGAAGGACCAGCTGGATAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGATTTC AAGGGCTACCTGGGCTGTCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTAATGCCA CAGGCCGAGAATCAGGACCCAGACATCAAGGCCCACGTGAACTCTCTGGGCGAGAATCTGAAGAC CCTGAGACTGAGGCTGAGACGCTGTCACAGATTCCTGCCATGCGAGAACAAGAGCAAGGCCGTGG AGCAGGTGAAGAACGCCTTCAATAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGTCTGAGTTC GACATCTTTATCAACTACATCGAGGCCTACATGACCATGAAGATCCGGAAC(SEQ ID NO:14)。

R0467VCLE gene sequence (699bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTGACATCCA GATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCATCGGCGACCGCGTGACCATCACCTGCCGCG CCAGCCAGGGCATCGACAACTGGCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG CTGATCTACGACGCCAGCAACCTGGACACCGGCGTGCCCAGCCGCTTCAGCGGCAGCGGCAGCGG CACCTACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACTTCGCCGTGTACTTCTGCCAGCA GGCCAAGGCCTTCCCCCCCACCTTCGGCGGCGGCACCAAGGTGGACATCAAGAGAACTGTGGCCG CTCCATCCGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAGAGTGGCACCGCCTCTGTGGTCTG TCTGCTGAACAATTTCTACCCCCGTGAGGCAAAGGTGCAGTGGAAAGTCGATAACGCCCTGCAGTC CGGAAATAGCCAGGAGTCTGTGACAGAACAGGACAGTAAGGATTCAACTTATTCTCTGTCTAGTAC CCTGACACTGTCTAAAGCTGACTACGAGAAGCACAAAGTGTATGCATGCGAAGTCACCCATCAGGG GCTGTCATCCCCTGTGACAAAGTCCTTTAATCGCGGTGAATGT(SEQ ID NO:15)。

R0468VCHE gene sequence (1395bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTGAGGTGC AGCTGGTGCAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGCGCCTGAGCTGCGCCGC CAGCGGCTTCACCTTCAGCAGCTACAGCATGAACTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGG AGTGGGTGAGCAGCATCAGCAGCAGCAGCAGCTACATCTACTACGCCGACAGCGTGAAGGGCCGC TTCACCATCAGCCGCGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGCGCCGA GGACACCGCCGTGTACTACTGCGCCCGCGTGACCGACGCCTTCGACATCTGGGGCCAGGGCACCAT GGTGACCGTGAGCAGCGCCTCTACCAAGGGACCCTCTGTGTTTCCTCTGGCTCCCTCCAGCAAGTC TACCTCTGGTGGAACAGCTGCCCTGGGCTGCCTGGTCAAGGATTACTTTCCTGAGCCTGTGACCGT GTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGG CCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGC AATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACA AGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGGCCGGCGCCCCCAGCGTGTTCCTGTTCC CCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGAC GTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACG CCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTG CTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGCAGCTGCCCG CCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTG CCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTA CCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC CCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCG CTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGAGCCTGAGCCTGAGCCCCGGCGCC(SEQ ID NO:16)。

R0523VCHE gene sequence (1956bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTGAGGTGC AGCTGGTGGAGAGCGGCGGCGGCCTGGGCCACCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGG CAGCGGCTTCACCTTCAGCAGCTACAGCATGCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGG AGTGGGTGAGCGCCATCGGCACCGGCGGCGGCACCTACTACGTGGACAGCGTGAAGGGCAGGTTC ACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGG ACATGGCCGTGTACTACTGCGCCAGGGACCTGGCCGCCGCCGGCAGGTACTACTACGGCATGGACG TGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGCCTCTACCAAGGGACCCTCTGTGTTTCCT CTGGCTCCCTCCAGCAAGTCTACCTCTGGTGGAACAGCTGCCCTGGGCTGCCTGGTCAAGGATTAC TTTCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCTGGCGTGCACACCTTTCCA GCTGTGCTGCAGTCCTCCGGCCTGTACTCTCTGTCCTCTGTCGTGACCGTGCCTTCCAGCTCTCTGG GCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAGAGTG GAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGGCCGGCGC CCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGCACCCCCGAGGT GACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGT GGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTG AGCAACAAGCAGCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCCGCG AGCCCCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAACCAGGTGAGCCTGACC TGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGA GAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGC TGACCGTGGACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGC CCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCGCCGGCGGAGGCGGCAGC GGTGGCGGTGGCAGCGGAGGCGGCGGCAGCGGAGGTGGAGGCAGCGGCTCCCCCGGCCAGGGCA CCCAGAGCGAGAACTCTTGTACCCACTTCCCCGGCAACCTGCCCAATATGCTGCGGGATCTGAGAG ACGCCTTCAGCAGAGTGAAGACCTTCTTTCAGATGAAGGACCAGCTGGATAACCTGCTGCTGAAG GAGAGCCTGCTGGAGGATTTCAAGGGCTACCTGGGCTGTCAGGCCCTGAGCGAGATGATCCAGTTC TACCTGGAGGAGGTAATGCCACAGGCCGAGAATCAGGACCCAGACATCAAGGCCCACGTGAACTC TCTGGGCGAGAATCTGAAGACCCTGAGACTGAGGCTGAGACGCTGTCACAGATTCCTGCCATGCG AGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAATAAGCTGCAGGAGAAGGGCAT CTACAAGGCCATGTCTGAGTTCGACATCTTTATCAACTACATCGAGGCCTACATGACCATGAAGATC CGGAAC(SEQ ID NO:17)。

R0523VCLE gene sequence (702bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTGAGATCGT GCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGG GCCAGCCAGAGCGTGAGCAGCACCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAG GCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCA GCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCC AGCAGTACGGCGGCAGCACCATCACCTTCGGCCAGGGCACCAGGCTGGAGATCAAGAGGACTGTG GCCGCTCCATCCGTCTTCATTTTTCCCCCTAGCGACGAACAGCTGAAGAGTGGCACCGCCTCTGTG GTCTGTCTGCTGAACAATTTCTACCCCCGTGAGGCAAAGGTGCAGTGGAAAGTCGATAACGCCCTG CAGTCCGGAAATAGCCAGGAGTCTGTGACAGAACAGGACAGTAAGGATTCAACTTATTCTCTGTCT AGTACCCTGACACTGTCTAAAGCTGACTACGAGAAGCACAAAGTGTATGCATGCGAAGTCACCCAT CAGGGGCTGTCATCCCCTGTGACAAAGTCCTTTAATCGCGGTGAATGT(SEQ ID NO:18)。

R0568 gene sequence (555bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTCACCACCA CCATCACCACTCCCCCGGCCAGGGCACCCAGAGCGAGAACTCTTGTACCCACTTCCCCGGCAACCT GCCCAATATGCTGCGGGATCTGAGAGACGCCTTCAGCAGAGTGAAGACCTTCTTTCAGATGAAGGA CCAGCTGGATAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGATTTCAAGGGCTACCTGGGCTGTC AGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTAATGCCACAGGCCGAGAATCAGGAC CCAGACATCAAGGCCCACGTGAACTCTCTGGGCGAGAATCTGAAGACCCTGAGACTGAGGCTGAG ACGCTGTCACAGATTCCTGCCATGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCT TCAATAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGTCTGAGTTCGACATCTTTATCAACTACAT CGAGGCCTACATGACCATGAAGATCCGGAAC(SEQ ID NO:19)。

R0595 Gene sequence (1257bp)

ATGGGATGGTCATGCATAATACTCTTTCTTGTGGCTACTGCTACCGGGGTTCACTCTGGCTCTGG CTCCGGCAGCGGCTCCGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCC CCGAGCTGGCCGGCGCCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCA GCCGCACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGCGAGGAGCAGTAC AACAGCACCTACCGCGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGA GTACAAGTGCAAGGTGAGCAACAAGCAGCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCA AGGGCCAGCCCCGCGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTGACCAAGAAC CAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAG CAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCT TCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGCTGGCAGCAGGGCAACGTGTTCAGCTGCAGC GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAGTC CCCCGGCCAGGGCACCCAGAGCGAGAACTCTTGTACCCACTTCCCCGGCAACCTGCCCAATATGCT GCGGGATCTGAGAGACGCCTTCAGCAGAGTGAAGACCTTCTTTCAGATGAAGGACCAGCTGGATA ACCTGCTGCTGAAGGAGAGCCTGCTGGAGGATTTCAAGGGCTACCTGGGCTGTCAGGCCCTGAGC GAGATGATCCAGTTCTACCTGGAGGAGGTAATGCCACAGGCCGAGAATCAGGACCCAGACATCAA GGCCCACGTGAACTCTCTGGGCGAGAATCTGAAGACCCTGAGACTGAGGCTGAGACGCTGTCACA GATTCCTGCCATGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAATAAGCTG CAGGAGAAGGGCATCTACAAGGCCATGTCTGAGTTCGACATCTTTATCAACTACATCGAGGCCTACA TGACCATGAAGATCCGGAAC(SEQ ID NO:20)

R0467VCHE amino acid sequence

MGWSCIILFLVATATGVHSEVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLE WVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKQLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGS GGGGSGGGGSGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQ VKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:21)。

R0467VCLE amino acid sequence

MGWSCIILFLVATATGVHSDIQMTQSPSSVSASIGDRVTITCRASQGIDNWLGWYQQKPGKAPKLLI YDASNLDTGVPSRFSGSGSGTYFTLTISSLQAEDFAVYFCQQAKAFPPTFGGGTKVDIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:11)。

R0468VCHE amino acid sequence

MGWSCIILFLVATATGVHSEVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLE WVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVTDAFDIWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKQLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGA(SEQ ID NO:10)。

R0523VCHE amino acid sequence

MGWSCIILFLVATATGVHSEVQLVESGGGLGHPGGSLRLSCAGSGFTFSSYSMHWVRQAPGKGLE WVSAIGTGGGTYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARDLAAAGRYYYGMDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELAGAPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKQLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAG GGGSGGGGSGGGGSGGGGSGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLL LKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:22)。

R0523VCLE amino acid sequence

MGWSCIILFLVATATGVHSEIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGGSTITFGQGTRLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:23)。

R0568 amino acid sequence

MGWSCIILFLVATATGVHSHHHHHHSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMK DQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRC HRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN(SEQ ID NO:13)。

R0595

MGWSCIILFLVATATGVHSGSGSGSGSEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKQLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSPGQGTQSENSC THFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQ AENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFI NYIEAYMTMKIRN(SEQ ID NO:24)

Experimental materials: PcdDNA^TM3.4 TOPO^TMTA Cloning Kit (Invitrogen A14697); 5-alpha Competent E.coli (compent E.coli) (NEB C2987I); DL2000 DNA Marker (TAKARA 3427A); q5 High Fidelity DHA Polymerase (High-Fidelity DNA Polymerase) (NEB M0491L); AxyPrep DNA Gel Extraction Kit (Gel Extraction Kit) (AxyGEN AP-GX-50); hind III (NEB); ecori (neb); t4 DNA ligase (TAKARA 2011A); endotoxin-free plasmid macroextract kit (TIANGEN DP 117);

the instrument equipment comprises: PCR instrument (Tprofessional TR 20); an electrophoresis apparatus (DYY-TC); gel imager (Smart Gel N); a centrifuge (H1650-W); micro thermostats (HW-8C); clean bench (SDJ series); a constant temperature oscillator (H2-9211K); constant temperature water bath (HH-4A)

The experimental method comprises the following steps: (1) gene synthesis

The amino acid sequences of R0467, R0468, R0523, R0568 and R0595 were respectively subjected to humanized base optimization, and their coding nucleotide sequences were artificially synthesized. The coding nucleic acid sequences of R0467, R0468, R0523, R0568 and R0595 are respectively shown in SEQ ID NO 14-15, SEQ ID NO 15-16, SEQ ID NO 17-18, SEQ ID NO 19 and SEQ ID NO 20.

(2) Vector selection

PCDNA3.4A vector is selected according to the purpose of plasmid to be constructed, the output of exogenous gene expression product, the preparation difficulty of vector DNA and the analysis result of enzyme cutting site of vector and target segment. The vector was purchased from Invitrogen corporation, and the constructed plasmid maps are shown in FIGS. 2 a-2 h.

(3) Primer design

The specific oligonucleotide primer sequences for amplifying R0467, R0468, R0523, R0568 and R0595 were finally determined by designing primers by SnapGene software (SnapGene Version 1.1.3) as shown in Table 1 below.

TABLE 1

(4) PCR amplification

Using a PCR instrument (Tprofessional TR20), using a Q5 High Fidelity DNA Polymerase (NEB M0491L) and using the synthesized sequences as templates and primers 1F/1R respectively; 2F/2R; 2F/3R was subjected to PCR amplification.

Total volume of reaction: 50 μ L of PCR 5 Xbuffer 10 μ L, High GC buffer 10 μ L, DNA template 0.5 μ L, Q5 High Fidelity DHA Polymerase (High-Fidelity DNA Polymerase)0.5 μ L, dNTP (25mM)4 μ L, and specific upper and lower primers (200 nmol/L final concentration) 1.5 μ L each, and sterile double distilled water was added to a total volume of 50 μ L;

reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min followed by subsequent denaturation at 94 ℃ for 30 sec, followed by annealing at 55 ℃ for 30 sec and extension at 72 ℃ for 1 min 30 sec for 30 cycles.

(5) Amplification product purification

The amplification product obtained in step (4) was subjected to electrophoresis using agarose gel (2% agarose gel) containing 2g of agarose per 100ml of gel by an electrophoresis apparatus (DYY-TC) to detect whether or not it specifically amplified the target fragment, using DL2000 DNAmarker (TAKARA 3427A) to generate a ladder. The result of observation by a Gel imager (Smart Gel N) shows that the PCR product is single band after electrophoresis, has the size of about 1.3Kb,1.4Kb and 800bp, has no impurity band, and indicates that the PCR product is single and has no non-specific amplification. And recovering the purified product after electrophoresis by using an Axygen agarose Gel recovery Kit (AxyPrep DNA Gel Extraction Kit, AxyGEN AP-GX-50) to obtain the target DNA molecule.

(6) Restriction of vectors and fragments of interest

Performing double enzyme digestion on the target DNA molecule and the vector molecule by Hind III (NEB) and EcoRI (NEB) to obtain corresponding cohesive ends, and performing enzyme digestion reaction in water bath at 37 ℃ for 1h (reaction system: Hind III 1 muL; EcoRI 1 muL; 10 XCutsmart Buffer 5 muL; plasmid/DNA 10 muL-12 muL is less than or equal to 1 mug, and the total volume is 50 muL (sterile water is supplemented to the total volume)); and recovering the large fragment of the recovered vector enzyme by using 1% agarose gel electrophoresis by using an Axygen agarose gel recovery kit, and directly recovering the enzyme fragment of the target gene by using an Axygen agarose gel recovery reagent.

(7) Ligation transformation

The large fragment recovered by digestion of the vector was ligated with the digested fragment of the target gene, and reacted at 14-16 ℃ for 4 hours using T4 DNA ligase (TAKARA 2011A) (reaction system: 0.5. mu.L of the digested fragment of the vector; 3.8. mu.L of the digested fragment of the target gene; 0.5. mu.L of T4 DNA ligase; 0.5. mu.L of 10 XT 4 DNA buffer). The whole ligation product was mixed with 100ul DH5 alpha competent bacteria (NEB C2987I) and incubated in ice for 30min, heat shocked at 42 ℃ for 90s, immediately transferred to ice and left for 2min, 450ul LB medium pre-warmed to room temperature was added, incubated at 37 ℃ for 60min at 250rpm in a constant temperature shaker (H2-9211K), the broth was centrifuged at 6000rpm for 3min, a portion of the supernatant was discarded, about 50. mu.l was mixed with a pipette and spread evenly on 100ug of ampicillin per ml of LB plate, and incubated in an inverted incubator (HW-8C) at 37 ℃ overnight.

(8) Picking monoclonal bacteria PCR verification

And selecting 9 single colonies, inoculating the single colonies into LB culture solution containing 100ug of ampicillin per milliliter, carrying out shaking culture at constant temperature of 37 ℃ at 250rpm for 5 hours, amplifying the bacterial solution by using corresponding amplification primers, selecting positive bacterial solution, and carrying out sequencing verification.

(9) Sequencing and result determination

Comparing the sequencing result with the designed sequence to obtain two target gene sequences and 4 isotype control sequences, and extracting plasmids by using an endotoxin-free plasmid large-extraction kit (TIANGEN DP 117).

EXAMPLE 2 preparation of proteins

The purpose is as follows: preparation of the protein of interest R0467 and control molecules R0568, R0595, R0523, R0511 (non-targeting monoclonal antibody, including Fab region and Fc region of antibody)

Experimental materials: expression cell line Expi293(Thermo Fisher Scientific; A14635), Dynamis medium (Gibco; A2617502), PEI (biosciences; MW40000), diabody (Gibco; 15140122), anticoagulant (Gibco; 0010057)

The instrument equipment comprises: cell culture shaking table (Adolf Kuhner; ISF4-XC), high-speed centrifugation (Hunan apparatus, H2050R), biological safety cabinet (ESCO AC2-4S1), CO₂Incubator (Japan Sanyo MCO-18AC), cell viability analyzer (Shanghai-Hengdk-8 AX)

The experimental method comprises the following steps:

1. expi293(ThermoFisher Scientific; A14635) cells one day prior to transient transfection were passaged by inoculating 1L shake flasks (conning; 431147) at a density of 2E6 with Dynamis medium (gibco; A2617502) and placed in a cell culture shaker (Adolf Kuhner; ISF4-XC) at 37 ℃; 8% CO₂(ii) a Culturing at 120 rpm.

2. On the day of transfection, Expi293 cells were counted using a cell counter (Countstar; IC1000), diluted with fresh DY to adjust the cell density to 2.9E 6; preparing for transfection; PEI: DNA 3: 1; adding the DNA-PEI mixture to Expi293 cells, mixing well, placing in a cell culture shaker (Adolf Kuhner; ISF4-XC) at 37 ℃; 8% CO₂(ii) a Culturing at 120 rpm.

3. The double antibody (gibco; 15140122) and the anticoagulant (gibco; 0010057) were added 4h after transfection.

4. Collecting samples after continuously culturing for 7 days, and firstly carrying out low speed 1000 rpm; 10 min; centrifuging at 4 deg.C (Hunan apparatus, H2050R), collecting cell supernatant, and centrifuging at high speed (Hunan apparatus, H2050R) at 12000 rpm; 30min 4 ℃; collecting the supernatant; and transferring to a purification group for purification.

The experimental results are as follows: cell supernatants were obtained and subjected to the following purification

Example 3 protein purification procedure

The purpose is as follows: separating and purifying from cell fermentation supernatant to obtain target Cyramza-h1.6Fc-IL10 fusion protein with SE-HPLC purity of more than 95%;

experimental materials: expi293 cell fermentation supernatant; buffer solution: and (3) buffer solution A: 1 × PBS (10mM PB,150mM NaCl, ph7.2, guangzhou chemical reagent factory, the following reagents all sourced from guangzhou chemical reagent factory unless otherwise specified); buffer B (20mM NaAc, pH3.4); CIP buffer (0.1M NaOH); neutralization buffer (1M Tris, pH8.0(aMRescO, 0497-500G)); buffer solution: 0.5M HCitrate (national drug, Hu test, 10007108); ultrafiltration concentration tubes (Millipore,

Ultra 15mL Centrifugal Filters，30KDa)

the instrument equipment comprises: protein A affinity chromatography packing (GE Healthcare, MabSelect SuRe LX), chromatography column (GE Healthcare, XK16/20, Column Volume (CV), 26 ml); a chromatography system: GE Healthcare, AKTA pure 150; portable pH meters (Horiba); microplate reader (Epoch, BioTek); centrifuge (xiang apparatus, H2050R).

The experimental method comprises the following steps:

a chromatography column (GE Healthcare, XK16/20, Column Volume (CV), 26ml) packed with Protein A affinity chromatography packing (GE Healthcare, MabSelect SuRe LX) was regenerated in a chromatography system (GE Healthcare, AKTA pure150), then equilibrated with buffer A for 10CV, the UV detector (UV Monitor) was reset, and the Expi293 cell fermentation supernatant was loaded in a bubble-sensitive manner. The column was washed with buffer A for 20CV, followed by stepwise elution with 100% buffer B for 7CV, and the UV-absorbing fractions of 20mAu to 20mAu at 280nm were collected, and-5% neutralization buffer was previously added to the collection tube to bring the final pH to 6.0 to 7.0, followed by washing in place (CIP, washing 3CV with Up-flow buffer for 3CV, holding for 3min, and then washing 5CV with Down-flow buffer A.

Performing affinity purification on supernatant obtained by centrifuging after cell fermentation according to the above process, adding 0.5M HCitrate to adjust pH to 6.0-7.5, standing at Room Temperature (RT) for more than 1 hour, filtering with 0.2um filter membrane, and analyzing purity of eluted sample after affinity chromatography by SE-HPLC (molecular sieve-high performance liquid chromatography). The purity results are shown in FIG. 3. Thus obtaining Cyramza-h1.6Fc-IL10 antibody fusion protein with the purity of more than 95%.

The experimental results are as follows: the desired various proteins of interest were obtained, wherein the SEC results for R0467 are shown in FIG. 3.

Example 4 determination of IL-10 terminal affinity by SRP

The purpose is as follows: detecting the affinity of the different proteins IL-10 and IL-10R

Experimental materials: from the fusion proteins h1.6Fc-wthIL10 (R0595B structure), iso-h1.6Fc-wthIL10 (R0523C structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), hIL10R his tag (ImmunoClone, ICR626Hu01), buffer system prepared in example 3: phosphate buffered saline (SBJ-0032, Senega) containing 0.02% Tween 20 (44112-: 10mM glycine (G8898-500G, Merck)

The instrument equipment comprises: molecular interaction analyzer ForteBIO (Octet OK)^e) (ii) a 96-well plates (Shanghai Jingan bioscience, J09602); AHC biosensor (Pall AHC) probe

The experimental method comprises the following steps: the fusion proteins R0467, R0595, R0523 to 5. mu.g/mL were diluted with 1. multidot. PBST buffer and solidified at this concentration to the molecular interaction analyzer ForteBIO (Octet OK^e) The receptor protein IL-10R corresponding to the IL-10 end of these proteins was diluted to 2. mu.g/mL with 1 × PBST buffer and diluted 2-fold down to 7 concentration gradients from the initial concentration on the AHC biosensor (Pall AHC) probe. Balancing the probes with PBST for 60s, binding to the receptors for 300s, respectively, thenAfter dissociation in PBST 480 s. After the probe was regeneratively neutralized in 10mM glycine, the next cycle of equilibration, binding, dissociation and regenerative neutralization was performed according to this method. All cycle speeds were set to 1000 rpm; the experimental temperature was 30 ℃.

The results are shown in Table 2.

Table 2: KD of IL-10 terminus of each molecule

Example 5 determination of the binding Activity of IL-10 terminal to IL1OR by ELISA

The purpose is as follows: comparing the binding ability of IL-10 terminal and IL-10R of the different proteins on protein level

Experimental materials: h1.6Fc-wthIL10 (R0595B structure), iso-h1.6Fc-wthIL10 (R0523C structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), GAH-IgG (Fc) -HRP (Sigma A0170), other color reagents for ELISA experiments and buffer were all laboratory-matched and omitted here.

The instrument equipment comprises: enzyme mark instrument (Molecular Devices spectramax i3x), plate washing machine (Shenzhen Shuhui Songku technology development Co., Ltd PW-960)

The experimental method comprises the following steps:

1. His-Tag Mouse mAb was diluted to 1. mu.g/mL (dilution: 50mM CB) one day in advance and plated at 100. mu.L/well overnight;

PBST washing the plate for three times, beating to dry, adding 200 mu L/hole blocking solution (1% BSA), and blocking for 1h at room temperature;

3. washing the plate once, then patting the plate dry, adding 100 mu L/well of diluted IL-10R (1 mu g/mL), and incubating for 1h at room temperature;

4. plates were washed 6 times and then patted dry, and each drug was diluted beforehand (100nM initial three-fold dilution 12 concentration points), added at 100. mu.L/well and incubated for 1h at room temperature.

5. The plate was washed 6 times and patted dry. The GAH-IgG Fc HRP (1:15000) was diluted and added at 100. mu.L/well and incubated at room temperature for 30 min.

6. After washing the plate for 6 times, the plate was patted dry, and then the developing solutions (solution A and solution B) were added thereto at a rate of 50. mu.L/well, respectively, and incubated at room temperature for 5 min. Add 50. mu.L/well stop solution and read at 450 nm.

The experimental results are as follows: the experimental result is shown in figure 4

In the samples that could be tested, R0467 was biologically similar to R0595, and R0523 was slightly inferior, but all on the same horizontal line.

Table 3: EC50 statistics for IL-10 end binding activity to IL-10R of each molecule

Sample(s)	EC50(nM)
		R0568	Since his tag was not detectable as in the experimental IL-10R his tag
R0595	0.1528
		R0523	0.2267
R0467	0.1893

Example 6 determination of the binding Activity of IL-10 terminus to CHO-IL10R by flow assay

The purpose is as follows: comparing the binding capacity of the IL-10 end of the different proteins to the IL-10R on the cellular level

Experimental materials: CHO-IL10R cells, wt hIL10 (R0568A structure), h1.6Fc-wthIL10 (R0595B structure), iso-h1.6Fc-wthIL10 (R0523C structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), APC anti-His tag (Biolegend 362605), APC anti-human IgG Fc (Biolegend 409306), BSA (Processary 9048-46-8), PBS solution as laboratory self-prepared solution, FCM buffer (3% PBS buffer solution for BSA is now prepared).

The instrument equipment comprises: centrifuge (Hunan instrument L-530), biological safety cabinet (ESCO AC2-4S1), and CO₂An incubator (MCO-18 AC, Japan), a cell viability analyzer (constant DK-8AX, Shanghai), a flow cytometer (Beckman Cytoflex),

The experimental method comprises the following steps:

1. taking out CHO-IL10R cells, centrifuging for 4min at 300g, and removing supernatant;

2. adjusting the cell density to 4E6 cells/ml, adding 96-well V-shaped plates according to the experimental design, 50ul cells/well;

3. antibody dilution: dilute the sample with FCM buffer (942.4nM, 3X dilution);

4. adding the diluted antibody into a 96-hole V-shaped plate according to the experimental design, wherein each hole is 50 mu L, in addition, 100 mu L of FCM buffer is required to be added for contrast, resuspending cells, putting the 96-hole plate at 4 ℃, and incubating for 30 min;

5. taking out a 96-well plate, centrifuging for 4min at 300g, carefully removing the supernatant, adding 200 mu L of FCM buffer per well, centrifuging for 4min at 300g again, and carefully removing the supernatant;

6. adding diluted APC anti-human IgG Fc (1:400) APC-GAHIS (1:400) according to the experimental design, incubating at 4 deg.C for 30min at 100 μ L/well;

7. taking out a 96-well plate, centrifuging for 4min at 300g, carefully removing the supernatant, adding 200 mu L of FCM buffer per well, centrifuging for 4min at 300g again, and carefully removing the supernatant;

8. resuspend with 1xPBS 100. mu.L/well and FCM detect.

The experimental results are as follows: the experimental result is illustrated in figure 5

Table 4: binding Activity of IL-10 terminal of each molecule to CHO-IL10R EC50 statistic

Sample(s)	EC50(nM)
		R0595	5.276
R0523	8.083
		R0467	5.899

Example 7 detection of biological Activity of IL-10 terminal by reporter Gene method

The purpose is as follows:

comparison of the relative biological Activity of the IL10 Ends of the molecules by reporter Gene method

Experimental materials: the fusion proteins wt hIL10 (R0568A structure), h1.6Fc-wthIL10 (R0595B structure), iso-h1.6Fc-wthIL10 (R0523C structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), hIL10 luciferase reporter cell line (Gimeran GM-C07927), Bright-Lumi luciferase reporter assay kit (Beyotime RG051M), DPBS (Hyclone SH3002802), DMEM (Gibco 11965-084), FBS (Gibco 11091-148), TE (Gibco 25200056), PS (Gibco 15140-122), Hygromycin B (Invitrogen 10687010),

Blastincidin(Solarbio B9300-10mg)、Puromysin(Solarbio B9300-10mg)

The instrument equipment comprises: enzyme labeling instrument (Molecular Devices spectramax i3x), centrifuge (Hunan Xiang instrument L-530), biological safety cabinet (ESCO AC2-4S1), and CO₂Incubator (Japan Sanyo MCO-18AC), cell viability analyzer (Shanghai-Hengdk-8 AX), horizontal vibration plate (Kylin-Bell MH-1)

The experimental method comprises the following steps:

1) r0568, R0595, R0523, R0467 were all diluted from a 4-fold gradient of 222nM (final concentration of 111nM) for 9 concentration gradients;

2) digestion of hIL with good growth status10 reporter gene cells (Gilles Palmata organism GM-C07927), counting and reselecting after medium washing, adjusting the cell density to 5 x10⁵One cell/mL (final density 2.5X 10)⁵one/mL);

3) adding each diluted molecular drug into a 96-hole white board at a rate of 50 muL/hole, and adding the cells with the adjusted density into the 96-hole white board at a rate of 50 muL/hole;

4)CO₂culturing in incubator (MCO-18 AC of Sanyo Japan) for 16 h;

5) adding 100 μ l of luciferase reporter gene detection reagent (Beyotime RG051M) into each well, oscillating the plate horizontally for 10min (Kylin-Bell MH-1) for reaction, detecting by an enzyme-labeling instrument (Molecular Devices spectramax i3x), and analyzing data.

The experimental results are as follows:

r0467 was slightly more (not significantly) biologically active as R0568, R0595, but R0523 was slightly less; the results are shown in FIG. 6.

Table 5: reporter gene method for detecting biological activity EC50 of each molecule in solution state

Sample(s)	EC50(nM)
		R0568	3.95
R0595	3.046
		R0523	5.108
R0467	3.005

Example 8 inhibition of U937 TNF-alpha production following LPS-induced activation

The purpose is as follows: 1. comparison of relative biological activity of each molecule for non-immobilized antibodies (not targeted to bind VEGFR 2);

2. the immobilized antibody (targeted to bind VEGFR2) R0467 was relatively biologically more active than the other control molecules.

Experimental materials: the fusion proteins wt hIL10 (R0568A structure), h1.6Fc-wthIL10 (R0595B structure), iso-h1.6Fc-wthIL10 (R0523C structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), DPBS (Hyclone SH3002802), RPMI 1640(Gibco 11875-085), FBS (Gibco 11091-148), PMA (Sigma P1585), LPS (Sigma L4516), Human TNF- α uncoated Elisa kit (Invitrogen 88-7346-88), anti-his tag mAb (Minn. Ex 1901), VEGFR2(Sino 10012-H08H) prepared in example III

The instrument equipment comprises: enzyme labeling instrument (Molecular Devices spectramax i3x), centrifuge (Hunan Xiang instrument L-530), biological safety cabinet (ESCO AC2-4S1), and CO₂Incubator (Japan Sanyo MCO-18AC), cell viability analyzer (Shanghai-Hengdk-8 AX)

The experimental method comprises the following steps:

1. non-immobilized antibody moiety

1) Preparing a 1640+ 10% FBS culture medium containing 10nM PMA;

2) taking a proper amount of cultured U937 cells, centrifuging for 4min at 300g, resuspending and counting by using a prepared culture medium, adjusting the density to 5E5 cells/mL, and adding the cells into a 6-well plate, wherein each well contains 4mL of the cells;

3) culturing at 37 deg.C with 5% CO2 for 24 h;

4) taking out the 6-hole plate, gently blowing up the cells at the bottom by using a pipette gun, and collecting the cells;

5) centrifuging at 300g for 5min, discarding the supernatant, and washing with 1640 culture medium for three times;

6) counting cells, and adjusting the cell density to 2E6 cells/mL;

7) dilution sample 83.33nM start, 4-fold gradient dilution, 10 gradient points;

8) adding the prepared cells into a 96-well plate, wherein each well has 50 ul;

9) adding the diluted sample into a 96-well plate according to the experimental design, adding 50ul of 1640+ 10% FBS culture medium into each well without adding an antibody well;

10) mixing, and incubating at 37 deg.C for 30 min;

11) preparing a culture solution containing LPS with the concentration of 10ng/ml by using a 1640+ 10% FBS culture medium, and adding 100ul of LPS into each hole according to the experimental design;

12) after further incubation at 37 ℃ for about 21-22h, the supernatant was taken for TNF-. alpha.factor detection (specific experimental procedures were followed by Invitrogen 88-7346-88 as described in the instructions of the test kit);

2. immobilized antibody moiety

1) Preparing a 1640+ 10% FBS culture medium containing 10nM PMA;

3) culturing at 37 deg.C with 5% CO2 for 24 h;

4) anti-his mAb antibody (1. mu.g/mL) was prepared in PBS and added to a 96-well flat bottom plate at 100. mu.l/well overnight at 4 ℃ according to the experimental design;

5) taking out the flat bottom plate, sucking out the supernatant, and adding 150 μ l PBS to wash twice;

6) VEGFR2 his tag was prepared in PBS, 1. mu.g/mL, added to a 96-well flat bottom plate according to the experimental design, 100. mu.l/well and incubated at 37 ℃ for 1.5 h;

7) taking out the 6-hole plate, gently blowing up the cells at the bottom by using a pipette gun, and collecting the cells;

8) centrifuging at 300g for 5min, discarding the supernatant, and washing with 1640 culture medium for three times;

9) counting cells, and adjusting the cell density to 2E6 cells/mL;

10) dilution sample 83.33nM start, 4-fold gradient dilution, 10 gradient points;

11) adding the prepared cells into a 96-well plate, wherein each well has 50 ul;

12) adding the diluted sample into a 96-well plate according to the experimental design, adding 50ul of 1640+ 10% FBS culture medium into each well without adding an antibody well;

13) mixing, and incubating at 37 deg.C for 30 min;

14) preparing a culture solution containing LPS with the concentration of 10ng/ml by using a 1640+ 10% FBS culture medium, and adding 100ul of LPS into each hole according to the experimental design;

15) after further incubation at 37 ℃ for about 21-22h, the supernatant was taken for TNF-. alpha.factor detection (specific experimental procedures were followed by Invitrogen 88-7346-88 as described in the instructions of the test kit);

the experimental results are as follows:

1. non-immobilized antibody moiety (see FIG. 7 for results)

Table 6: inhibition of the functional Activity of U937 molecules IC50 (non-immobilized)

Sample(s)	IC50(nM)
		R0568	0.06655
R0595	0.07445
		R0523	0.09246
R0467	0.129

2. Immobilized antibody moiety (see FIGS. 8a-8c for results)

Table 7: inhibition of the functional Activity of U937 molecules IC50 (immobilization)

Example 9 Cyramza end affinity determination by SRP

The purpose is as follows: detecting the affinity of the Cyramza end of the different proteins and VEGFR2

Experimental materials: from the fusion proteins prepared in example three, Cyrmaza-h1.6Fc (R0468D structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), Cyramza (LILLY), VEGFR2(Sino 10012-H08H), buffer system: phosphate buffered saline (SBJ-0032, Senega) containing 0.02% Tween 20 (44112-: 10mM glycine (G8898-500G, Merck)

The instrument equipment comprises: molecular interaction analyzer ForteBIO (Octet OK)^e) (ii) a 96-well plates (Shanghai Jingan bioscience, J09602); Ni-NTAbiosensor (Pall Ni-NTA) probe

The experimental method comprises the following steps: VEGFR2 was diluted to 3. mu.g/mL with 1 × PBST buffer and solidified at this concentration into the molecular interaction analyzer ForteBIO (Octet OK^e) On the Ni-NTAbiosensor (Pall Ni-NTA) probe of (1). Each protein to be detected was then diluted to 10 μ g/mL with 1 × PBST buffer and diluted 2-fold down to 7 concentration gradients from this initial concentration. Probes 60s were equilibrated with PBST, bound to receptors 300s, respectively, and then dissociated in PBST for 480 s. After the probe was regeneratively neutralized in 10mM glycine, the next cycle of equilibration, binding, dissociation and regenerative neutralization was performed according to this method. All cycle speeds were set to 1000 rpm; the experimental temperature was 30 ℃.

The experimental results are as follows:

table 8: k at the Cyramza end of each molecule_D

Sample(s)	K_on(1/Ms)	K_off(1/s)	K_D(M)
				R0468	1.33×10⁵	<1.0×10^-7	<1.0×10^-12
R0467	1.14×10⁵	<1.0×10^-7	<1.0×10^-12
				Cyramza	1.15×10⁵	<1.0×10^-7	<1.0×10^-12

Example 10 determination of binding Activity of Cyramza terminus to VEGFR2 by ELISA

The purpose is as follows: comparison of Cyramza-IL10 antibody fusion proteins with Cyramza for binding Activity differential to VEGFR2

Experimental materials: the fusion proteins Cyrmaza-h1.6Fc (R0468D structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), Cyramza (LILLY), VEGFR2(Sino 10012-H08H), BSA (Biotech 9048-46-8), His-Tag Mouse mAb (Minn. Ex. Bio, MY1901), GAH-IgG (GAH-IgG) (Fab-HRP (Sigma A9917), other chromogenic reagents used in ELISA experiments, and buffer were prepared by laboratory and omitted here.

The experimental method comprises the following steps:

3. after washing the plate once, patting dry, 100. mu.L/well of diluted VEGFR2 (0.5. mu.g/mL) was added and incubated for 1h at room temperature;

4. plates were washed 6 times and then patted dry, and each drug was diluted beforehand (12 concentration points at 300nM initial three-fold dilution) and added at 100. mu.L/well and incubated for 1h at room temperature.

5. The plate was washed 6 times and patted dry. GAH-IgG Fab HRP (1:15000) was diluted, added at 100. mu.L/well and incubated at room temperature for 30 min.

The experimental results are as follows: the results are shown in FIG. 9

R0467 and R0468 showed comparable binding activity to VEGFR 2.

Table 9: EC50 values for binding Activity of Cyramza end of each molecule to VEGFR2

Sample(s)	EC50(nM)
		R0468	0.2310
R0467	0.2525
		Cyramza	0.2431

Example 11 measurement of the biological Activity of Cyramza end-blocked VEGFR2 and VEGF165 by ELISA

The purpose is as follows: comparison of the Cyramza-IL10 antibody fusion protein and the Cyramza Activity differential in blocking the binding of VEGFR2 to VEGF165

Experimental materials: the fusion proteins Cyrmaza-h1.6Fc (R0468D structure), Cyrmaza-h1.6Fc-wthIL10 (R0467E structure), VEGFR2(Sino 10012-H02H), VEGF165(Acro VE5-H5248-50ug), BSA (Production 9048-46-8), Anti-his tag HRP (Biolegend 652504), other chromogenic reagents and buffer used in ELISA experiments were laboratory-matched and were omitted.

The experimental method comprises the following steps:

1. VEGFR2(Fc tag) was diluted to 2. mu.g/mL (dilution: 50mM CB) in the afternoon of the previous day and plated overnight at 100. mu.L/well;

2. the plate was washed 3 times in the morning the following day and patted dry. Adding 200. mu.L/non-pore blocking solution (1% BSA), blocking for 1h at room temperature;

3. the plate was washed 1 time and blotted dry, and the samples were diluted (300nM starting three-fold dilution of 12 concentration points) and added at 100. mu.L/well and incubated for 1h at room temperature.

4. hVEGF165-his (1. mu.g/mL) was diluted and added at 100. mu.L/well and incubated at room temperature for 1 h.

5. The microplate was washed 5 times and patted dry, and anti-his HRP (1:5000) was diluted and added at 100. mu.L/well and incubated at room temperature for 30 min.

6. Washing the enzyme labeling plate for 6 times, drying, adding developing solutions (solution A and solution B), adding according to 50 μ L/hole, respectively, and incubating at room temperature for 10 min.

7. Add 50. mu.L/well stop solution and read at 450 nm.

The experimental results are as follows: the results are shown in FIG. 10

Table 10: IC50 value of each molecule Cyramza end blocking VEGF165 and VEGFR2

Sample(s)	IC50(nM)
		R0468	0.4168
R0467	0.4379
		Cyramza	0.3874

Example 12 in vivo anti-tumor evaluation of R0467 fusion proteins

Pharmacodynamic evaluation of R0467 in B16-F1 mouse melanoma subcutaneous transplantation model

The purpose is as follows: the anti-tumor efficacy of the bifunctional protein Cyrmaza-h1.6Fc-wthIL10 (R0467E structure) was tested in a B16-F1 mouse melanoma subcutaneous transplantation model, and the bifunctional protein Cyrmaza-h1.6Fc-wthIL10 (R0467E structure) was tested for its bifunctional effects on angiogenesis and immune activation, as compared to the isotype control antibody iso-h1.6Fc (R0511), anti-VEGFR2 antibody (Cyramza) alone, IL-10-Fc fusion protein (R0356) alone, and a combination thereof. The experiment was performed twice, the first experiment using the marketed antibody Cyramza and the Fc fusion protein of IL-10 (R0356) as a unilateral functional control; in the second experiment, Cyramza-h1.6Fc (R0468D structure) and iso-h1.6Fc-wthIL10 (R0523C structure) which are modified from Cyramza subtype are used as unilateral functional controls.

Experimental materials: human KDR transgenic C57BL/6 mice, female, 6-8 weeks (shanghai south model biotechnology limited); B16-F1 tumor cells (national experimental cell resource sharing platform, resource number: 3111C0001CCC 000365). DMEM medium (Gibco, cat # 11965092), FBS (Gibco,10091-

The instrument equipment comprises: electronic balance (Shanghai Shunhui scientific instruments Co., Ltd., JA12002), slide caliper (Shanghai Meinaite practice Co., Ltd., MNT-150T), microscope (Chongqing Ote optical instruments Co., Ltd., BDS200), medical centrifuge (Hunan Xiang instruments laboratory development Co., Ltd., L530R), digital display thermostatic water bath (Puruis mechanical Co., Ltd., HH-S), carbon dioxide incubator (Pingsu health medical instruments Co., Ltd., MCO-18AC), double vertical super clean bench (Sn-free easy cleaning equipment Co., SW-CJ-VS2)

Experimental methods

Cell culture: mouse melanoma cells (B16-F1) were cultured in DMEM medium (Gibco) containing 10% fbs (Gibco), 1% glutamine and 1% penicillin-streptomycin.

Inoculation: B16-F1 cells were collected in the logarithmic growth phase and the cell concentration was adjusted to 2X 106/mL. (1) 20 female C57BL/6 mice were inoculated subcutaneously with B16-F1 cells in a volume of 0.1 mL/mouse, i.e., 2X 105/mouse.

Administration: the day of vaccination was recorded as day 0 (D0), mice were randomized into 5 groups of 7 mice by body weight, dosing was started (dosing schedule see table 11 below), and the doses of each group were converted on an equimolar basis.

Table 11: protocol design (first experiment):

after inoculation, animals will be checked daily for morbidity and mortality. After the start of treatment, tumor major and minor diameters were measured 2 times a week. According to the formula: (1/2) x major axis x minor axis, tumor volume is calculated, and animal tumor growth and effects of treatment on normal behavior, such as mobility, food and water consumption, weight gain/loss, eye/hair/skin, etc., are examined and recorded.When each mouse reached the end of the experiment (mice lost more than 20% weight or tumor volume more than 2000 mm)³To the kindness endpoint), mice were euthanized.

TABLE 12 evaluation of the antitumor Activity of R0467 in the B16-F1 model (first experiment, mean tumor volume mm³)

The experimental results are as follows: under equimolar dosage, the tumor inhibition effect of R0467 is obviously better than that of the anti-VEGFR2 antibody Cyrmaza which is already on the market and Fc fusion protein R0356 of IL-10, and a remarkable bifunctional effect is exerted (figure 11).

To further confirm that R0467 has dual effects of anti-angiogenesis and immune activation, a second experiment was performed in a B16-F1 mouse melanoma model using R0468 engineered from the Cyrmaza subtype and the isotype control fusion protein R0523 of whole antibody with IL-10 as controls. Experimental materials, instruments, model set-up and grouping methods were the same as for the first experiment, with D0 on the day of inoculation and the dosing schedule shown in table 13.

Table 13: experimental protocol design (second experiment):

after inoculation, animals will be checked daily for morbidity and mortality. After the start of treatment, tumor major and minor diameters were measured 2 times a week. Data calculation methods were as described in the first experiment. The results of mean tumor volume after inoculation are shown in table 14 and fig. 12.

Table 14: evaluation of antitumor Activity of R0467 in B16-F1 model (second experiment, mean tumor volume mm 3):

days after implantation	R0511	R0468	R0467	R0523+R0468
					8	21.07	14.34	1.93	5.25
12	104.68	33.07	4.29	5.79
					15	213.46	63.46	3.60	25.06
19	881.82	222.07	4.45	115.35
					21	1361.38	435.20	16.42	197.19
23	1973.46	787.33	37.80	354.39
					25	2613.81	1233.89	77.39	596.99

The experimental results are as follows: at equimolar doses, the tumor suppression effect of R0467 was clearly superior to that of the anti-VEGFR2 antibody R0468 and to that of the combination of R0468 and IL-10 fusion protein R0523 (FIG. 12).

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A fusion protein, comprising: a VEGF antagonist antibody comprising a Fab region and an Fc region, and IL-10.

2. The fusion protein of claim 1, wherein the VEGF antagonist antibody comprises at least one selected from the group consisting of anti-VEGF antibodies and anti-VEGFRs antibodies;

optionally, the VEGF comprises at least one selected from VEGF-A, VEGF-B, PlGF, VEGF-A, VEGF-B, VEGF-E, PlGF-2, VEGF-A, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGF-A, VEGF-C, PlGF-2, VEGF-C, VEGF-D;

optionally, the VEGFRs include at least one selected from VEGFR1, NRP-1, VEGFR2, NRP-2, VEGFR 3;

optionally, the anti-VEGFRs antibody is an anti-VEGFR2 antibody;

optionally, the anti-VEGFRs antibody specifically binds to the extracellular region of VEGFR.

3. The fusion protein of claim 2, wherein the anti-VEGFR antibody comprises a CDR sequence selected from at least one of the following or an amino acid sequence at least 95% identical thereto:

heavy chain variable region CDR sequences: 1 to 3 of SEQ ID NO,

light chain variable region CDR sequences: 4-6 of SEQ IN NO;

optionally, the anti-VEGFRs antibody comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs 1, 2 and 3, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs 1, 2 and 3, and light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs 4, 5 and 6, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs 4, 5 and 6;

optionally, the anti-VEGFRs antibody has the amino acid sequence as set forth in SEQ ID NO: 7;

optionally, the anti-VEGFRs antibody has the amino acid sequence as set forth in SEQ ID NO: 8 in a light chain variable region.

4. The fusion protein of claim 2, wherein the anti-VEGFRs antibody comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of the at least one of a heavy chain constant region and a light chain constant region being derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof;

optionally, the light chain constant region and the heavy chain constant region of the anti-VEGFRs antibody are both from a human IgG antibody or a mutant thereof;

optionally, the light chain constant region and the heavy chain constant region of the anti-VEGFRs antibody are both from human IgG 1.

5. The fusion protein of claim 1, wherein the Fc region has the a327Q, G237A, and L235A mutations;

optionally, the Fc region further has the K447A mutation;

optionally, the Fc region has the amino acid sequence shown in SEQ ID NO 9.

6. The fusion protein of claim 2, wherein the anti-VEGFRs antibody has a heavy chain having the amino acid sequence shown in SEQ ID No. 10 and a light chain having the amino acid sequence shown in SEQ ID No. 11.

7. The fusion protein of claim 1, further comprising a linker peptide, wherein the N-terminus of the linker peptide is linked to the C-terminus of the VEGF antagonist antibody, and wherein the C-terminus of the linker peptide is linked to the N-terminus of the IL-10;

optionally, the amino acid sequence of the connecting peptide has the amino acid sequence shown in SEQ ID NO. 12.

8. A fusion protein, comprising: a targeting antibody comprising a Fab region and an Fc region having the a327Q, G237A and L235A mutations, and IL-10.

9. The fusion protein of claim 8, wherein the targeting antibody is an antibody targeting VEGF or VEGFRs;

optionally, the VEGFR includes at least one selected from the group consisting of VEGFR1, NRP-1, VEGFR2, NRP-2, VEGFR 3;

optionally, the targeting antibody is a VEGFR2 targeting antibody;

optionally, the targeting antibody specifically binds to the extracellular region of VEGFR.

10. The fusion protein of claim 9, wherein the targeting antibody comprises a CDR sequence or an amino acid sequence at least 95% identical thereto selected from at least one of:

heavy chain variable region CDR sequences: 1 to 3 of SEQ ID NO,

light chain variable region CDR sequences: 4-6 of SEQ IN NO;

optionally, the targeting antibody comprises heavy chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs 1, 2 and 3, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs 1, 2 and 3, and light chain variable region CDR1, CDR2, CDR3 sequences as set forth in SEQ ID NOs 4, 5 and 6, respectively, or amino acid sequences at least 95% identical to SEQ ID NOs 4, 5 and 6;

optionally, the targeting antibody has the amino acid sequence as set forth in SEQ ID NO: 7;

optionally, the targeting antibody has the amino acid sequence as set forth in SEQ ID NO: 8 in a light chain variable region.

11. The fusion protein of claim 8, wherein the targeting antibody comprises a heavy chain constant region comprising the Fc region and a light chain constant region, at least a portion of the heavy chain constant region and the light chain constant region being derived from at least one of a murine antibody, a human antibody, a primate antibody, or mutant thereof;

optionally, the light chain constant region and the heavy chain constant region of the targeting antibody are both from a human IgG antibody;

optionally, the light chain constant region and the heavy chain constant region of the targeting antibody are both from human IgG 1.

12. The fusion protein of claim 8, wherein the Fc region further has the K447A mutation;

optionally, the Fc region has the amino acid sequence shown in SEQ ID NO 9.

13. The fusion protein of claim 9, wherein the targeting antibody has a heavy chain having the amino acid sequence set forth in SEQ ID No. 10 and a light chain having the amino acid sequence set forth in SEQ ID No. 11.

14. The fusion protein of claim 8, further comprising a linker peptide, wherein the N-terminus of the linker peptide is linked to the C-terminus of the targeting antibody, and wherein the C-terminus of the linker peptide is linked to the N-terminus of the IL-10;

15. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 14.

16. The nucleic acid molecule of claim 15, wherein said nucleic acid has the nucleotide sequence set forth in SEQ ID NO. 14 or 15.

17. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 14, the nucleic acid molecule of claim 15 or 16.

18. The pharmaceutical composition of claim 17, further comprising a pharmaceutically acceptable carrier.

19. Use of the fusion protein of any one of claims 1 to 14, the nucleic acid molecule of claim 15 or 16 or the pharmaceutical composition of claim 17 or 18 for the preparation of a medicament for the treatment or prevention of a vascular proliferative disease, an autoimmune disease or a tumor.