CN103965363B - Fusion protein efficiently combined with PD-1 and VEGF, coding sequence and application thereof - Google Patents

Abstract

The invention belongs to the field of biomedicine, and relates to a fusion protein efficiently combined with PD-1 and VEGF, a coding sequence and application thereof. Specifically, the invention relates to an isolated fusion protein, which comprises the following peptide fragments: (1) an extracellular domain peptide of human PD-L2; (2) the peptide fragment of 2 nd immunoglobulin-like structural domain of human FLT-1 extracellular region or/and the peptide fragment of 3 rd immunoglobulin-like structural domain of KDR extracellular region; and optionally (3) an Fc fragment of a human immunoglobulin. The fusion protein can inhibit the growth of tumor cells, promote the death of the tumor cells, inhibit the inactivation of T cells and enhance the tumor killing capability.

Description

Fusion protein efficiently combined with PD-1 and VEGF, coding sequence and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to a fusion protein efficiently combined with PD-1 and VEGF, a coding sequence and application thereof.

Background

The monoclonal antibody has definite target, good clinical application safety and obvious curative effect, and at present, 10 tumor monoclonal antibodies are approved by the United states FDA to be sold on the market and become first-line medicaments for treating various malignant tumors. On the one hand, however, the full-length antibody has a large molecular weight, is not easy to enter solid tumor tissues, and is difficult to achieve an effective treatment concentration in tumors, so that an anti-tumor effect is achieved. On the other hand, the tumor pathogenesis is complex, the signal paths in cells are redundant or crossed, the curative effect of the single target is insufficient, and the drug resistance is easy to form. However, the combined treatment by applying a plurality of monoclonal antibodies aiming at different targets has better curative effect than single target treatment, but the cost is very expensive, and the patients are difficult to bear. Therefore, it is very necessary to develop a multi-target antibody protein drug with small molecular weight.

Programmed death molecule 1 (PD-1) is a T cell natural immunosuppressive molecule, and tumor cells can act on the surface PD-1 of the T cells by over-expressing ligands (PD-L1/PD-L2) of the PD-1 on the cell surface, activate a PD-1 pathway, induce the apoptosis of the T cells and form immune escape and tolerance. Thus, PD-1 has become an important target for tumor therapy. A phase I clinical trial (296 patients) of a PD-1 antibody (BMS-936558 or MDX-1106) shows that the PD-1 antibody can enable one fourth to one fifth of patients with non-small cell lung cancer, melanoma or renal cell carcinoma to generate objective responses (J Clin Oncol.2010;28: 3167-75; N Engl J Med.2012;366: 2443-54), and the antibody has good clinical application value.

VEGF has the function of promoting angiogenesis, tumor cells can secrete VEGF in a large quantity, and the VEGF acts on VEGFR 1(Vascular endothelial growth factor receptor 1, also called FLT-1) and VEGFR2 (Vascular endothelial growth factor receptor 2, also called KDR) receptors to induce the generation of new blood vessels in tumor tissues and rapidly expand the tumor volume (Nat Med.1999;5: 1359-64); in addition, VEGF can inhibit the differentiation and maturation of DCs, suppress the anti-tumor T cell immune response of the body and evade immune surveillance (blood. 2003;101: 4878-86). Currently, the monoclonal antibody bevacizumab (avastin) against VEGF has been approved by the FDA in the united states for marketing, and is widely used in the treatment of colon cancer, breast cancer, non-small cell lung cancer, kidney cancer, glioma, with a sale of $ 64.6 billion in 2010.

Interestingly, the extracellular domain polypeptides of Flt-1 or KDR also block the VEGF signaling pathway in vitro, inhibiting the growth of vascular endothelial cells (Cancer Res.2002;9: 633-40). Fusion of Flt-1 to certain specific immunoglobulin-like (Ig-like) domains in the extracellular domain of KDR (e.g., Flt-1's 2 Ig-like domain Flt1-D2, KDR's 3 Ig-like domain KDR-D3) is effective in blocking cellular VEGF signaling pathways by genetic engineering techniques (Proc Natl Acad Sci USA.2002;99: 11393-8; Gene Ther.2009;16: 10-6).

Therefore, if the tumor cell VEGF and T cell PD-1 pathway can be blocked simultaneously, the tumor growth can be inhibited effectively, the immune tolerance can be eliminated, and the anti-tumor effect can be exerted.

Disclosure of Invention

The inventor obtains a fusion protein through intensive research and creative work, and surprisingly discovers that the fusion protein can be efficiently combined with PD-1 and VEGF, simultaneously blocks tumor cell VEGF and T cell PD-1 access, has obvious anti-tumor effect and has the potential of being applied to the preparation of medicaments for preventing and treating malignant tumors or gene therapy medicaments. The following invention is thus provided:

one aspect of the present invention relates to an isolated fusion protein comprising the following peptide fragments:

1) an extracellular domain peptide of human PD-L2;

2) the peptide fragment of 2 nd immunoglobulin-like structural domain of human FLT-1 extracellular region or/and the peptide fragment of 3 rd immunoglobulin-like structural domain of KDR extracellular region; and optionally

3) Fc fragment of human immunoglobulin.

Without being bound by theory, the extracellular domain peptide of human PD-L2 is capable of efficiently binding to PD-1.

Without being bound by theory, in nature, both PD-1 and PDL1/PDL2 are membrane proteins whose binding involves the response of tumor cells and immune cells, acting as suppressors of T cells. The designed fusion protein is in a free state and has a similar action mechanism with an antibody. Although it can also bind to PD-1 through the extracellular region of PD-L2, the function is to competitively bind to PD-1 on the surface of T cells with PDL1/PDL2 on the surface of tumor cells, block the function of PD-1, which is the natural immune molecule of T cells, and prevent the apoptosis of T cells.

Without being limited by theory, the peptide segment of 2 nd immunoglobulin-like domain of extracellular region of VEGF receptor FLT-1 (abbreviated as Flt 1-D2) and the peptide segment of 3 rd immunoglobulin-like domain of KDR extracellular region (abbreviated as KDR-D3) can both efficiently bind VEGF.

The fusion protein of any one of the present invention, wherein the extracellular domain peptide fragment of human PD-L2 comprises or is SEQ ID NO: 1.

The fusion protein of any one of the invention, wherein the peptide fragment of immunoglobulin-like domain 2 of the extracellular region of human FLT-1 comprises or is SEQ ID NO: 3.

The fusion protein of any one of the invention, wherein the peptide fragment of the 3 rd immunoglobulin-like domain of the human KDR extracellular region comprises or is SEQ ID NO: 5.

The fusion protein according to any one of the present invention, wherein the Fc fragment is located on the side of the fusion protein near the C-terminus, relative to the peptide fragments of (1) and (2).

In one embodiment of the present invention, the sequence of linkage from the N-terminus to the C-terminus of the fusion protein is the polypeptide described in (1), the polypeptide described in (2), and the Fc fragment described in (3), in that order.

In one embodiment of the present invention, the sequence of linkage from the N-terminus to the C-terminus of the fusion protein is the polypeptide described in (2), the polypeptide described in (1), and the Fc fragment described in (3), in that order.

In the present invention, the Fc region of the human immunoglobulin includes not only the native Fc region sequence, but also a sequence that is artificially modified, altered or mutated and has the function of the Fc region. The fusion protein according to any one of the invention, wherein the Fc segment is selected from the full-length Fc segment of human immunoglobulin IgG subtypes (such as IgG1, IgG2, IgG3, or IgG 4), IgM, or IgA; preferably, the Fc fragment does not have ADCC effect; preferably, the Fc fragment is the full-length Fc fragment of IgG 4.

Without being bound by theory, one of the targets of the present invention is PD-1 on T cells, which only requires blocking the PD-1 pathway of T cells, preventing their apoptosis, and IgG Fc with ADCC effect cannot be selected. Otherwise, when the fusion protein binds to T cells, the body will clear the T cells by ADCC effect, whereas IgG4Fc has no ADCC effect and is therefore preferred.

The fusion protein of any one of the invention, wherein the extracellular region peptide fragment of human PD-L2 is in one or more copies; specifically, two copies.

Without being bound by theory, multiple copies may enhance affinity for a target, but if the number of copies is too large, correct folding of the spatial conformation of the fusion protein may be affected in the art, thereby affecting its function; specifically, 2 copies.

The fusion protein according to any one of the invention, wherein the peptide fragment of immunoglobulin-like domain 2 of human FLT-1 extracellular region or/and the peptide fragment of immunoglobulin-like domain 3 of KDR extracellular region are in one or more copies; specifically, two copies.

The fusion protein according to any one of the present invention, wherein, when in multiple copies,

the peptide fragment of the extracellular region of human PD-L2, the peptide fragment of the 2 nd immunoglobulin-like domain of the extracellular region of human FLT-1 or/and the peptide fragment of the 3 rd immunoglobulin-like domain of the extracellular region of KDR are homologous tandem repeats or interval tandem repeats.

The fusion protein according to any one of the present invention, further comprising a signal peptide; preferably, the amino acid sequence of the signal peptide is as set forth in SEQ ID NO: shown at 9.

Without being bound by theory, the signal peptide may enhance the secretion of the fusion protein and may eventually be cleaved by the protease after the signal peptide is expressed along with other amino acid sequences of the fusion protein. The protease has a certain recognition sequence, and the signal peptide is fused with the peptide segment behind the signal peptide to form a new amino acid sequence, so that if the selected signal peptide is not proper, the protease can be cut by mistake, and the fusion protein is inactivated. Thus, preferably, the amino acid sequence of the signal peptide is as set forth in SEQ ID NO: shown at 9.

The fusion protein according to any one of the invention, wherein the peptide fragment of the extracellular region of human PD-L2, the peptide fragment of the 2 nd immunoglobulin-like domain of the extracellular region of human FLT-1 and/or the peptide fragment of the 3 rd immunoglobulin-like domain of the extracellular region of KDR, the Fc fragment and the optional signal peptide are directly connected or connected through 1 or more same or different linkers (Linker sequences); preferably, the amino acid sequence of the Linker is shown in SEQ ID NO: 11 or SEQ ID NO: shown at 13.

The fusion protein is a protein product which is obtained by connecting the coding regions of two or more genes end to end through a DNA recombination technology and has multiple functions through the same gene expression frame. Without being bound by theory, a key issue in constructing fusion proteins is the Linker sequence (Linker), i.e., Linker peptide, between two or even more proteins of different origin. Its length is important for the folding and stability of the protein. If the linker sequence is too short, it may affect the folding of the higher structure of the two proteins (peptides), thereby interfering with each other and inactivating one or more of the proteins of origin; if the linker sequence is too long, problems with immunogenicity are involved, since the linker sequence itself is a new antigen and as the amino acid sequence of the fusion protein is extended, the difficulty of expression increases and the overall folding may present new problems. Thus, specific reference to each protein, due to their different respective conformations, requires specific analysis of how they are fused together without affecting the function of each moiety. Unfortunately, although the reliance on the primary structure of a protein to predict its secondary structure has produced significant progress, the understanding of the relationship between sequence and structure is limited and the secondary structure of the fused protein cannot be accurately modeled by software. This current situation directly leads to the current lack of reliable selection criteria for the design of linker peptide sequences.

Without being limited by theory, the inventor increases the expression quantity of the fusion protein by selecting proper signal peptide and linker, and is beneficial to obtaining the fusion protein with better space conformation, thereby improving the activity of the fusion protein.

In a specific embodiment, the fusion protein is represented schematically in figure 1. They are obtained by constructing the encoding DNA of PD-L2 extracellular region, Flt-1, KDR and human IgG4Fc segment through gene engineering technology. Wherein Sp1 and Sp2 are abbreviations of Linker1 and Linker2 respectively. The fusion proteins are designated PVP1, PVP2, PVP3 and PVP4, respectively, in the present invention. Wherein:

the PVP1 is formed by connecting PD-L2, Flt1-D2 and human IgG4Fc segments in sequence through a protein Linker.

The PVP2 is formed by sequentially connecting Flt1-D2, PD-L2 and human IgG4Fc segments through a protein Linker.

The PVP3 is formed by connecting PD-L2, KDR-D3 and human IgG4Fc segments in sequence through a protein Linker.

The PVP4 is formed by sequentially connecting KDR-D3, PD-L2 and human IgG4Fc segments through a protein Linker.

The fusion protein according to any one of the present invention, wherein the amino acid sequence thereof comprises or is SEQ ID NO: 15 to SEQ ID NO: 18, or a sequence shown in fig. 18.

Yet another aspect of the present invention relates to a method for preparing the fusion protein of the present invention, comprising the steps of:

a. synthesizing an expression frame according to the amino acid sequence and the coding sequence of each peptide segment of the fusion protein;

b. inserting the expression frame obtained in the step a into a proper vector, transforming the expression frame into a proper host cell, and extracting and purifying plasmids;

c. transfecting the purified plasmid to a proper cell, culturing and amplifying the transfected cell, collecting the supernatant of the culture, and purifying to obtain a fusion protein;

preferably, the suitable vector in step b is selected from the group consisting of a plasmid, a recombinant virus (e.g., a recombinant adenovirus or a recombinant adeno-associated virus), a phage, preferably wherein the plasmid is a eukaryotic expression plasmid or a prokaryotic expression plasmid, such as pcdna3.1, pDC315 or pAAV-MCS;

preferably, the suitable host cell described in step b is a bacterium or a fungus, such as e.coli (e.g. DH5 a);

preferably, the suitable cells described in step c are eukaryotic expression host cells, such as 293 cells (embryonic kidney cells, available from the american type collection, ATCC).

In a particular embodiment, the method particularly comprises the steps of:

a. synthesizing an expression frame according to the amino acid sequence and the coding sequence of each peptide segment of the fusion protein;

b. inserting the expression frame obtained in the step a into the EcoRI site of pCDNA3.1 (+) vector, transforming to E.coli (DH 5 alpha), extracting and purifying plasmid;

c. and (3) transfecting the purified plasmid to 293 cells, culturing and amplifying the transfected cells, collecting culture supernatant, and purifying to obtain the fusion protein.

In a specific example, in step c, the plasmid obtained in step b is transfected into 293cells using Lipofectamine 2000.

In a specific embodiment, in step c, after transfection, preferably 1 to 4 days after transfection, and further preferably 2 or 3 days after transfection, the 293 cells after transfection are transferred to a DMEM medium with neomycin, and the cells are cloned by a limiting dilution method.

In a specific example, the cloned cells are selected to establish a cell line stably transfected with a corresponding expression vector and having neomycin resistance. Then, the stably transfected cells were extensively expanded by shake flask culture, and culture supernatants were collected. Preferably, the screening is carried out for 18 to 24 days, preferably 19 to 22 days, and more preferably 21 days.

In a specific embodiment, step c, wherein said purification is performed by gel filtration affinity chromatography.

Another aspect of the invention relates to an isolated polynucleotide encoding a fusion protein according to any of the invention.

A polynucleotide according to any one of the invention comprising or being SEQ ID NO: 19 to SEQ ID NO: 22 or a degenerate sequence thereof.

Yet another aspect of the present invention relates to a recombinant vector comprising a polynucleotide according to any one of the present invention; specifically, the vector is a plasmid, a recombinant adenovirus, a recombinant adeno-associated virus or a bacteriophage; preferably, the plasmid is a eukaryotic expression plasmid or a prokaryotic expression plasmid, such as pCDNA3.1, pDC315, pAAV-MCS.

Yet another aspect of the invention relates to a recombinant host cell comprising a recombinant vector according to any one of the invention; in particular, the host cell is a bacterial (e.g., e.coli), fungal or eukaryotic expression host cell (e.g., 293 cell).

Yet another aspect of the invention relates to a composition comprising a fusion protein according to any of the invention or a polynucleotide according to any of the invention or a recombinant vector according to any of the invention or a recombinant host cell according to any of the invention; optionally, it further comprises a pharmaceutically acceptable carrier or excipient.

A further aspect of the invention relates to the use of a fusion protein according to any of the invention or a polynucleotide according to any of the invention or a recombinant vector according to any of the invention or a recombinant host cell according to any of the invention or a composition according to any of the invention for the preparation of an anti-tumor drug or a transgenic therapeutic drug; specifically, the tumor is one or more selected from lung cancer, liver cancer, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer and prostate cancer; more specifically, the liver cancer is liver cancer transplantation tumor, and the ovarian cancer is ovarian cancer transplantation tumor.

A method for treating and/or preventing and/or adjunctively treating a tumor, comprising the step of administering to a subject an effective amount of a fusion protein, polynucleotide, recombinant vector, recombinant cell or composition according to any of the invention; specifically, the tumor is one or more selected from lung cancer, liver cancer, lymphoma, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct cancer, gallbladder cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer and prostate cancer; more specifically, the liver cancer is liver cancer transplantation tumor, and the ovarian cancer is ovarian cancer transplantation tumor.

The dosage to be administered will depend on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, and the condition and past medical history of the patient being treated.

The following gives an explanation of some of the terms to which the present invention relates:

the term "PD-1" refers to human programmed cell death factor 1(programmed cell death 1), the official ID number of the NCBI Genbank is 5133, and the corresponding protein sequence number is NP-005009.1.

The term "VEGF" refers to human vascular growth factor A (vascular endothelial growth factor A), NCBI official abbreviation VEGFA, gene ID number 7422, corresponding protein numbers NP _001020537.2, NP _003367.4, NP _001020538.2, NP _001020539.2, NP _001020540.2, NP _001020541.2, NP _001028928.1, NP _001165093.1, NP _001165094.1, NP _001165095.1, NP _001165096.1, NP _001165097.1, NP _001165098.1, NP _001165099.1, NP _001165100.1, NP _001165101.1, NP _001191313.1, NP _ 001191314.1.

The term "Flt-1" refers to human vascular growth factor receptor 1, the NCBI group is generally referred to as fms-related systemic kinase 1(vascular endothelial growth factor/vascular permeability factor receptor), also abbreviated VEGFR1, with the gene ID number 2321 and the corresponding protein numbers NP-002010.2, NP-001153392.1, NP-001153502.1, NP-001153503.1.

The term "extracellular region" refers to a segment of a membrane protein that is located outside of a cell.

The term "domain" refers to a region of a protein biomacromolecule having a specific structure and independent function, the number of amino acid residues in a common domain is between 100 and 400, the smallest domain has only 40-50 amino acid residues, and the larger domain can exceed 400 amino acid residues.

The term "immunoglobulin-like domain" means that the domain can form a steric structure similar to an immunoglobulin.

The term "homologous tandem repeat" refers to a polypeptide in which one amino acid residue is repeated 1 or more times.

The term "spacer tandem repeat" refers to a polypeptide in which one amino acid residue is connected to another amino acid residue, or a unit thereof is repeated more than 1 time.

The term "Linker" refers to a polypeptide that functions to make two adjacent peptide sequences do not interfere with each other spatially.

The term "degeneracy" refers to the phenomenon that the same amino acid has two or more codons.

The invention relates to a 'high-efficiency binding PD-1 and VEGF' which means that the affinity constant Kd is less than 9.9 multiplied by 10^-7mol/L。

The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The term "composition" may also refer to a pharmaceutical composition that may be used to effect treatment, prevention, alleviation and/or alleviation of a disease or disorder described herein in a subject.

The term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or amelioration of a disease or disorder described herein in a subject.

The term "subject" can refer to a patient or other animal, particularly a mammal, e.g., a human, mouse, dog, monkey, cow, horse, etc., that receives a composition of the invention to treat, prevent, ameliorate, and/or alleviate a disease or disorder described herein.

Advantageous effects of the invention

The invention fuses the polypeptide with high-efficiency binding PD-1 ability with the polypeptide with high-efficiency binding VEGF ability, so that the fusion protein can be simultaneously bound with PD-1 and VEGF, and simultaneously seals two channels of VEGF activated by tumor cells and PD-1 activated by T cells, thereby inhibiting the growth of tumor cells, promoting the death of the tumor cells, inhibiting the inactivation of the T cells and enhancing the tumor killing ability. Meanwhile, the Fc peptide segment of the antibody is fused, which is beneficial to prolonging the in vivo half-life of the fusion protein, increasing the antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity) of the antibody and improving the anti-tumor effect of the fusion protein.

Accordingly, because tumor cells PD-L1/PD-L2 and VEGF are over-expressed, by utilizing a similar strategy, a polypeptide which can effectively bind to PD1 ligand (a peptide segment of PD-1 protein which can bind to the ligand, such as PD1 extracellular region), a polypeptide which can effectively bind to VEGF (Flt-1 domain peptide segment orKDR domain 3 peptide segment) and a human IgG1 Fc segment are fused, can act on the tumor cells, block the pathways of tumor cell VEGF and PD-L1/PD-L2, inhibit the angiogenesis and immune tolerance of tumor cells and play an anti-tumor role.

Drawings

FIG. 1: schematic structural diagrams of fusion proteins PVP1, PVP2, PVP3 and PVP 4. They are obtained by constructing the coding DNA of PD-L2, Flt1-D2, KDR-D3 and human IgG4Fc segment by genetic engineering technology. L1, L2 are abbreviations for Linker1 and Linker2, respectively.

FIG. 2: and (3) determining the affinity constant of the fusion protein PVP1 and the PD-1 extracellular region. The assay curves of recombinant human PD-1 were 1000nM, 500nM, 250nM, 125nM, 62.5nM, and 0nM, respectively, from top to bottom.

FIG. 3: the affinity constant of the fusion protein PVP1 with VEGF was determined. The assay curves for recombinant human VEGF protein were 1000nM, 500nM, 250nM, 125nM, 62.5nM and 0nM, respectively, from top to bottom.

FIG. 4: the affinity of the fusion protein PVP1 to activated T cells was determined. A, anti-PD-1 antibody (positive control); b, PVP1, C, anti-CD 3 antibody (T cell marker), D, blank control.

FIG. 5: the statistical result of the curative effect of the plasmid carrying the coding sequence of the fusion protein (PVP 1, PVP 3) for treating the liver cancer Hep3B transplantation tumor in the body of the mouse.

Detailed Description

Embodiments of the present invention will be described in detail with reference to 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 do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: design and synthesis of fusion protein expression frame and construction of expression vector

Splicing the whole fused amino acid sequence and coding DNA expression frame according to the amino acid sequence and coding sequence of each component of the fusion protein, wherein:

the amino acid residue sequence of the PD-L2 extracellular region is as follows:

LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVEN DTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWD YKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNV SVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLA SIDLQSQMEPRTHPTW(SEQ ID NO：1)

the coding sequence of the PD-L2 extracellular region is as follows:

CTCTTTACTGTGACCGTGCCAAAAGAACTGTATATCATTGAG CACGGGTCCAATGTGACCCTCGAATGTAACTTTGACACCGGCAGC CACGTTAACCTGGGGGCCATCACTGCCAGCTTGCAAAAAGTTGAA AACGACACTTCACCTCACCGGGAGAGGGCAACCCTCTTGGAGGA GCAACTGCCATTGGGGAAGGCCTCCTTTCATATCCCTCAGGTGCA GGTTCGGGATGAGGGACAGTACCAGTGCATTATTATCTACGGCG TGGCTTGGGATTACAAGTATCTGACCCTGAAGGTGAAAGCGTCCT ATCGGAAAATTAACACTCACATTCTTAAGGTGCCAGAGACGGACG AGGTGGAACTGACATGCCAAGCCACCGGCTACCCGTTGGCAGAG GTCAGCTGGCCCAACGTGAGCGTACCTGCTAACACTTCTCATTCT AGGACACCCGAGGGCCTCTACCAGGTTACATCCGTGCTCCGCCT CAAACCGCCCCCAGGCCGGAATTTTAGTTGCGTGTTTTGGAATAC CCACGTGCGAGAGCTGACTCTTGCATCTATTGATCTGCAGTCCCA GATGGAGCCACGGACTCATCCAACTTGG(SEQ ID NO：2)

the amino acid residue sequences of Flt1-D2 are:

GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLI PDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT (SEQ ID NO：3)

the coding sequence for Flt1-D2 is:

GGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTA TACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTA CGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACAC TTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGG CTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACC TGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCA CACATCGACAAACC(SE Q ID NO：4)

the amino acid residue sequence of KDR-D3 is:

SVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQ SGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVH (SEQ ID NO：5)

the coding sequence of KDR-D3 is:

TCTGTTGGAGAAAAGCTTGTCTTAAATTGTACAGCAAGAACTG AACTAAATGTGGGGATTGACTTCAACTGGGAATACCCTTCTTCGAA GCATCAGCATAAGAAACTTGTAAACCGAGACCTAAAAACCCAGTCT GGGAGTGAGATGAAGAAATTTTTGAGCACCTTAACTATAGATGGTG TAACCCGGAGTGACCAAGGATTGTACACCTGTGCAGCATCCAGTG GGCTGATGACCAAGAAGAACAGCACATTTGTCAGGGTCCAT(SEQ ID NO：6)

the amino acid residue sequence of human IgG4Fc is:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVD KRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：7)

the coding sequence for human IgG4Fc is:

GCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCT CCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAG CTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAG CAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCC ATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGT CTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGG ACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAG CCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGA ATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATAA(SEQ ID NO：8)

the amino acid residue sequence of the signal peptide is:

MEFWLSWVFLVAILKGVQC(SEQ ID NO：9)

the signal peptide coding sequence is:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA AAGGTGTCCAGTGT(SEQ ID NO：10)

the amino acid residue sequence of Linker1 is:

GGGGSGGGGSGGGGS(SEQ ID NO：11)

the Linker1 coding sequence is:

GGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGG GTCG(SEQ ID NO：12)

the amino acid residue sequence of Linker2 is:

GGGGGGGGG(SEQ ID NO：13)

the Linker2 coding sequence is:

GGTGGAGGTGGAGGTGGAGGTGGAGGT(SEQ ID NO：14)

the fusion protein PVP1 is formed by signal peptide-PD-L2 extracellular region-Linker 1-Flt1D2-Linker2-Fc fusion in sequence, and the amino acid sequence is as follows:

MEFWLSWVFLVAILKGVQCLFTVTVPKELYIIEHGSNVTLECNFD TGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQ VRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVEL TCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRN FSCVFWNTHVRELTLASIDLQSQMEPRTHPTWGGGGSGGGGSGGGG SGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPD GKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTGG GGGGGGGASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO：15)

the fusion protein PVP2 is formed by signal peptide-Flt 1D2-Linker1-PD-L2 extracellular region-Linker 2-Fc fusion in sequence, and the amino acid sequence is as follows:

MEFWLSWVFLVAILKGVQCGRPFVEMYSEIPEIIHMTEGRELVIP CRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTC EATVNGHLYKTNYLTHRQTGGGGSGGGGSGGGGSLFTVTVPKELYII EHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQL PLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINT HILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQV TSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWGG GGGGGGGASTKGPSVFPLAPC SRSTSE STAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK (SEQ ID NO：16)

the fusion protein PVP3 is formed by fusing a signal peptide-PD-L2 extracellular region-Linker 1-KDR-D3-Linker2-Fc in sequence, and the amino acid sequence is as follows:

MEFWLSWVFLVAILKGVQCLFTVTVPKELYIIEHGSNVTLECNFD TGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQ VRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVEL TCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRN FSCVFWNTHVRELTLASIDLQSQMEPRTHPTWGGGGSGGGGSGGGG SSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSG SEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHGGGG GGGGGASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：17)

the fusion protein PVP4 is formed by signal peptide-KDR-D3-Linker 1-PD-L2 extracellular region-Linker 2-Fc fusion in sequence, and the amino acid sequence is as follows:

MEFWLSWVFLVAILKGVQCSVGEKLVLNCTARTELNVGIDFNWE YPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCA ASSGLMTKKNSTFVRVHGGGGSGGGGSGGGGSLFTVTVPKELYIIEH GSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPL GKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHI LKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTS VLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWGGGG GGGGGASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：18)

the DNA coding sequence of PVP1 (SEQ ID NO: 19) and PVP2 (SEQ ID NO: 20) were delegated to Jielii

The entire expression cassette was synthesized by bioengineering (Shanghai) Ltd, inserted into the EcoRI site of pCDNA3.1 (+) vector (Invitrogen) (according to the instructions), transformed into E.coli (DH 5. alpha.) and sequenced correctly, and used as a plasmid by QiagenAnd extracting and purifying plasmids by using the plasmid purification kit to obtain high-quality plasmids of each recombinant expression vector. Each fusion protein coding sequence is as follows:

PVP1 coding sequence:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA AAGGTGTCCAGTGTCTCTTTACTGTGACCGTGCCAAAAGAACTGTA TATCATTGAGCACGGGTCCAATGTGACCCTCGAATGTAACTTTGAC ACCGGCAGCCACGTTAACCTGGGGGCCATCACTGCCAGCTTGCAA AAAGTTGAAAACGACACTTCACCTCACCGGGAGAGGGCAACCCTC TTGGAGGAGCAACTGCCATTGGGGAAGGCCTCCTTTCATATCCCTC AGGTGCAGGTTCGGGATGAGGGACAGTACCAGTGCATTATTATCT ACGGCGTGGCTTGGGATTACAAGTATCTGACCCTGAAGGTGAAAG CGTCCTATCGGAAAATTAACACTCACATTCTTAAGGTGCCAGAGAC GGACGAGGTGGAACTGACATGCCAAGCCACCGGCTACCCGTTGGC AGAGGTCAGCTGGCCCAACGTGAGCGTACCTGCTAACACTTCTCAT TCTAGGACACCCGAGGGCCTCTACCAGGTTACATCCGTGCTCCGC CTCAAACCGCCCCCAGGCCGGAATTTTAGTTGCGTGTTTTGGAATA CCCACGTGCGAGAGCTGACTCTTGCATCTATTGATCTGCAGTCCCA GATGGAGCCACGGACTCATCCAACTTGGGGTGGAGGCGGTTCAGG CGGAGGTGGCAGCGGCGGTGGCGGGTCGGGTAGACCTTTCGTAGA GATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAG GGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTT ACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAAC GCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAAC GTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGG GCATTTGTATAAGACAAACTATCTCACACATCGACAAACCGGTGGA GGTGGAGGTGGAGGTGGAGGTGCTTCCACCAAGGGCCCATCCGTC TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCC GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG TGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCA ACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG AGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTT CCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGT GGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGT GGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCC CAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGA GCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCT GGGTAAATGATAA(SEQ ID NO：19)

PVP2 coding sequence:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA AAGGTGTCCAGTGTGGTAGACCTTTCGTAGAGATGTACAGTGAAAT CCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCC TGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTC CACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAG TAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGG CTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAA ACTATCTCACACATCGACAAACCGGTGGAGGCGGTTCAGGCGGAG GTGGCAGCGGCGGTGGCGGGTCGCTCTTTACTGTGACCGTGCCAA AAGAACTGTATATCATTGAGCACGGGTCCAATGTGACCCTCGAATG TAACTTTGACACCGGCAGCCACGTTAACCTGGGGGCCATCACTGC CAGCTTGCAAAAAGTTGAAAACGACACTTCACCTCACCGGGAGAG GGCAACCCTCTTGGAGGAGCAACTGCCATTGGGGAAGGCCTCCTT TCATATCCCTCAGGTGCAGGTTCGGGATGAGGGACAGTACCAGTG CATTATTATCTACGGCGTGGCTTGGGATTACAAGTATCTGACCCTG AAGGTGAAAGCGTCCTATCGGAAAATTAACACTCACATTCTTAAGG TGCCAGAGACGGACGAGGTGGAACTGACATGCCAAGCCACCGGCT ACCCGTTGGCAGAGGTCAGCTGGCCCAACGTGAGCGTACCTGCTA ACACTTCTCATTCTAGGACACCCGAGGGCCTCTACCAGGTTACATC CGTGCTCCGCCTCAAACCGCCCCCAGGCCGGAATTTTAGTTGCGT GTTTTGGAATACCCACGTGCGAGAGCTGACTCTTGCATCTATTGAT CTGCAGTCCCAGATGGAGCCACGGACTCATCCAACTTGGGGTGGA GGTGGAGGTGGAGGTGGAGGTGCTTCCACCAAGGGCCCATCCGTC TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCC GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG TGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCA ACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG AGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTT CCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGT GGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGT GGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCC CAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGA GCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATG AGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCT GGGTAAATGATAA(SEQ ID NO：20)

PVP3 coding sequence:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA AAGGTGTCCAGTGTCTCTTTACTGTGACCGTGCCAAAAGAACTGTA TATCATTGAGCACGGGTCCAATGTGACCCTCGAATGTAACTTTGAC ACCGGCAGCCACGTTAACCTGGGGGCCATCACTGCCAGCTTGCAA AAAGTTGAAAACGACACTTCACCTCACCGGGAGAGGGCAACCCTC TTGGAGGAGCAACTGCCATTGGGGAAGGCCTCCTTTCATATCCCTC AGGTGCAGGTTCGGGATGAGGGACAGTACCAGTGCATTATTATCT ACGGCGTGGCTTGGGATTACAAGTATCTGACCCTGAAGGTGAAAG CGTCCTATCGGAAAATTAACACTCACATTCTTAAGGTGCCAGAGAC GGACGAGGTGGAACTGACATGCCAAGCCACCGGCTACCCGTTGGC AGAGGTCAGCTGGCCCAACGTGAGCGTACCTGCTAACACTTCTCAT TCTAGGACACCCGAGGGCCTCTACCAGGTTACATCCGTGCTCCGC CTCAAACCGCCCCCAGGCCGGAATTTTAGTTGCGTGTTTTGGAATA CCCACGTGCGAGAGCTGACTCTTGCATCTATTGATCTGCAGTCCCA GATGGAGCCACGGACTCATCCAACTTGGGGTGGAGGCGGTTCAGG CGGAGGTGGCAGCGGCGGTGGCGGGTCGTCTGTTGGAGAAAAGCT TGTCTTAAATTGTACAGCAAGAACTGAACTAAATGTGGGGATTGAC TTCAACTGGGAATACCCTTCTTCGAAGCATCAGCATAAGAAACTTG TAAACCGAGACCTAAAAACCCAGTCTGGGAGTGAGATGAAGAAAT TTTTGAGCACCTTAACTATAGATGGTGTAACCCGGAGTGACCAAGG ATTGTACACCTGTGCAGCATCCAGTGGGCTGATGACCAAGAAGAA CAGCACATTTGTCAGGGTCCATGGTGGAGGTGGAGGTGGAGGTGG AGGTGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTG CTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGT CAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC CTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCC CAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCC CCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATC AGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAA GACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTG CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCG TCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACC AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAG GGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATAA(SE Q ID NO：21)

PVP4 coding sequence:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA AAGGTGTCCAGTGTTCTGTTGGAGAAAAGCTTGTCTTAAATTGTAC AGCAAGAACTGAACTAAATGTGGGGATTGACTTCAACTGGGAATA CCCTTCTTCGAAGCATCAGCATAAGAAACTTGTAAACCGAGACCTA AAAACCCAGTCTGGGAGTGAGATGAAGAAATTTTTGAGCACCTTAA CTATAGATGGTGTAACCCGGAGTGACCAAGGATTGTACACCTGTG CAGCATCCAGTGGGCTGATGACCAAGAAGAACAGCACATTTGTCA GGGTCCATGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGT GGCGGGTCGCTCTTTACTGTGACCGTGCCAAAAGAACTGTATATCA TTGAGCACGGGTCCAATGTGACCCTCGAATGTAACTTTGACACCGG CAGCCACGTTAACCTGGGGGCCATCACTGCCAGCTTGCAAAAAGT TGAAAACGACACTTCACCTCACCGGGAGAGGGCAACCCTCTTGGA GGAGCAACTGCCATTGGGGAAGGCCTCCTTTCATATCCCTCAGGT GCAGGTTCGGGATGAGGGACAGTACCAGTGCATTATTATCTACGG CGTGGCTTGGGATTACAAGTATCTGACCCTGAAGGTGAAAGCGTC CTATCGGAAAATTAACACTCACATTCTTAAGGTGCCAGAGACGGAC GAGGTGGAACTGACATGCCAAGCCACCGGCTACCCGTTGGCAGAG GTCAGCTGGCCCAACGTGAGCGTACCTGCTAACACTTCTCATTCTA GGACACCCGAGGGCCTCTACCAGGTTACATCCGTGCTCCGCCTCA AACCGCCCCCAGGCCGGAATTTTAGTTGCGTGTTTTGGAATACCCA CGTGCGAGAGCTGACTCTTGCATCTATTGATCTGCAGTCCCAGATG GAGCCACGGACTCATCCAACTTGGGGTGGAGGTGGAGGTGGAGGT GGAGGTGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCC TGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTG GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGC CCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC CCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCAT CAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTC CCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGA AGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGT GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT GAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCC GTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGAC CAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCC CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT CTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGA GGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGATAA(SEQ ID NO：22)。

example 2: expression and purification of fusion proteins

High-quality plasmids of each recombinant expression vector obtained in example 1 were constructed and purified, and transfected into 293 cells (embryonic kidney cells, purchased from american type collection, ATCC) using Lipofectamine2000 (Invitrogen). After 2 days, the transfected 293 cells were transferred to a DMEM medium with neomycin, and the cells were cloned by a limiting dilution method. After 21 days of selection, cell strains with neomycin resistance and stably transfected corresponding expression vectors are established. Then, a large number of stably transfected cells were expanded by shake flask culture, and the culture supernatants were collected and purified for each fusion protein by gel filtration affinity chromatography. The molecular weights of PVP1, PVP2, PVP3 and PVP4 are 73.3KD, 72.7KD and 72.7KD respectively, and the concentration of the purified fusion protein is determined by an ELISA method.

Example 3: determination of affinity constants of fusion protein and PD-1 and VEGF

The fusion proteins PVP1 prepared in example 2 were added to 96-well plates (10. mu.g/ml, 200. mu.l) and loaded on Anti-Human IgG Fc Capture biosensors (Octet), and then the affinity thereof was measured with a biomacromolecule interactor (Octet Red 96) to recombinant Human PD-1 and VEGF proteins (200. mu.l, SinoBiological lnc.) at different concentrations (1000 nM, 500nM, 250nM, 125nM, 62.5nM, 0 nM), respectively, and the affinity constants Kd of PVP1 to PD-1 and VEGF, respectively, were 97.3nM (see FIG. 2) and 30.2nM (see FIG. 3). As known to those skilled in the artWith a general Kd of less than 10^-7(i.e., 100nM, smaller represents higher affinity), indicating that the two proteins are efficiently bound. This affinity is, of course, low relative to the affinity of the antibody for the antigen, but is sufficiently high to allow for binding between the natural receptor and the ligand.

Example 4: affinity of the fusion protein PVP1 to activated T cells

Taking peripheral blood mononuclear cells (1 × 10) of healthy people⁶Adding 10% fetal calf serum (HyQ, USA) into 1640 culture medium (GiBCo), adding gamma IFN (Phmingen, USA) 1000U/ml on day 1, and adding anti-human CD3 monoclonal antibody (Phmingen, USA) 20 μ g/L and IL-1 (R-1) after culturing for 24 hr&Company D) of 500U/ml and rhlL-2 (Beijing four-ring pharmaceutical factory) of 1000U/ml, and the liquid is changed every 3 days while rhlL-21000U/ml is added, and the activated CIK cells are obtained after 14 days.

600. mu.l of activated CIK cells were taken, and1X 10 cells in total⁶Each cell; centrifuging at 2000r/min for 4min, discarding the supernatant, and washing with 200 μ l PBS for 2 times; fusion protein PVP1 and control antibodies (anti-PD 1 antibody, anti-CD 3 antibody, purchased from R)&Company D) with 10% BSA at 1: 100 mixing, 100 mul/well, incubating for 70 min; centrifuging at 2000r/min for 4min, discarding the supernatant, and washing with 200 μ l PBS for 2 times; murine anti-human IgG4, FITC antibody with 10% BSA at 1: 100 mixing, 100 ul/hole, and incubating for 45 min; add 400. mu.l of physiological saline to each well and detect fluorescence by up-flow. The results are shown in FIGS. 4A-D.

The results showed that cultured CIK cells expressed the T cell marker CD3 for the most part (90.3%, see fig. 4C). Compared to the blank control (0%, see fig. 4D), the fusion protein PVP1 was able to bind efficiently to CIK cells (37.1%, see fig. 4B), although the detection rate was somewhat lower than the positive control (anti-PD-1 antibody, 82.7%, see fig. 4B).

Example 5: plasmid carrying fusion protein coding sequence for treating mouse liver cancer Hep3B transplantation tumor

Using Entrans^TMIn vivo transfection kit (Engreen Biosystem Co, Ltd.) for preparing and encapsulating the fusion proteins PVP1, PVP3 expression obtained in example 1The plasmid pCDNA3.1-PVP1, pCDNA3.1-PVP3, and the nanoparticle solution of the empty vector pCDNA3.1 (as per the procedure), a final 150. mu.l of nanoparticle solution contained 50. mu.g of plasmid.

4-5 weeks old nude mice were subcutaneously inoculated with1X 10 cell line of Hep3B (purchased from ATCC) of liver cancer⁷. Three weeks later, tumor-bearing mice were given a single injection of the nano-plasmid DNA solutions (150. mu.l each) encapsulating pCDNA3.1-PVP1, pCDNA3.1-PVP3 into the caudal vein, and the control group was injected with the same volume of nano-plasmid DNA encapsulating pCDNA3.1, and the change of the tumor volume with time was counted. As a result, it was found that the tumor volume increased more than 4-fold after 3 weeks in the control group, while the size of the tumor did not significantly change in the treated group (see FIG. 5).

Although specific embodiments of the invention have been described in detail. As will be appreciated by those skilled in the art. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (24)

Translated fromChinese

1.一种分离的融合蛋白，其由如下肽段组成：1. an isolated fusion protein, it is made up of following peptide segment:1)人类PD-L2的胞外区肽段；1) The extracellular domain peptide of human PD-L2;2)人类FLT-1胞外区第2个免疫球蛋白样结构域的肽段或/和KDR胞外区第3个免疫球蛋白样结构域的肽段；以及2) a peptide segment of the second immunoglobulin-like domain of the extracellular region of human FLT-1 or/and a peptide segment of the third immunoglobulin-like domain of the extracellular region of KDR; and3)人类免疫球蛋白的Fc段；3) Fc segment of human immunoglobulin;其中：in:所述人类PD-L2的胞外区肽段的氨基酸序列如SEQ ID NO：1所示；The amino acid sequence of the extracellular region peptide of the human PD-L2 is shown in SEQ ID NO: 1;所述人类FLT-1胞外区第2个免疫球蛋白样结构域的肽段的氨基酸序列如SEQ ID NO：3所示；The amino acid sequence of the peptide segment of the second immunoglobulin-like domain in the extracellular region of human FLT-1 is shown in SEQ ID NO: 3;所述人类KDR胞外区第3个免疫球蛋白样结构域的肽段的氨基酸序列如SEQ ID NO：5所示；The amino acid sequence of the peptide segment of the third immunoglobulin-like domain of the human KDR extracellular region is shown in SEQ ID NO: 5;所述的Fc段不具有ADCC效应；The Fc segment has no ADCC effect;相对于1)和2)中所述的肽段，所述Fc段位于融合蛋白的靠近C末端的一侧；Relative to the peptide fragments described in 1) and 2), the Fc fragment is located on the side close to the C-terminus of the fusion protein;所述人类PD-L2的胞外区肽段、人类FLT-1胞外区第2个免疫球蛋白样结构域的肽段和KDR胞外区第3个免疫球蛋白样结构域的肽段分别独立地为一个或两个拷贝。The peptide segment of the extracellular region of human PD-L2, the peptide segment of the second immunoglobulin-like domain of the extracellular region of human FLT-1, and the peptide segment of the third immunoglobulin-like domain of the extracellular region of KDR, respectively One or two copies independently.2.根据权利要求1所述的融合蛋白，其特征在于如下的(1)－(4)中的任一项或者多项：2. fusion protein according to claim 1 is characterized in that any one or more in following (1)-(4):(1)所述人类免疫球蛋白Fc段选自人类免疫球蛋白IgG各亚型、IgM或者IgA的全长Fc段；(1) The human immunoglobulin Fc segment is selected from the full-length Fc segment of each subtype of human immunoglobulin IgG, IgM or IgA;(2)当为两个拷贝时，所述人类PD-L2的胞外区肽段、人类FLT-1胞外区第2个免疫球蛋白样结构域的肽段和KDR胞外区第3个免疫球蛋白样结构域的肽段为同源串联重复或间隔串联重复；(2) When there are two copies, the peptide of the extracellular region of human PD-L2, the peptide of the second immunoglobulin-like domain of the extracellular region of human FLT-1 and the third peptide of the extracellular region of KDR The peptides of the immunoglobulin-like domain are homologous tandem repeats or spaced tandem repeats;(3)所述融合蛋白还包含信号肽；(3) the fusion protein further comprises a signal peptide;(4)所述人类PD-L2的胞外区肽段、人类FLT-1胞外区第2个免疫球蛋白样结构域的肽段和/或KDR胞外区第3个免疫球蛋白样结构域的肽段、人类免疫球蛋白的Fc段以及任选的信号肽之间为直接相连或通过1个或多个相同或者不同的Linker相连。(4) The peptide segment of the extracellular region of human PD-L2, the peptide segment of the second immunoglobulin-like domain of the extracellular region of human FLT-1 and/or the third immunoglobulin-like structure of the extracellular region of KDR The peptide segment of the domain, the Fc segment of human immunoglobulin and the optional signal peptide are connected directly or through one or more identical or different Linkers.3.根据权利要求2所述的融合蛋白，其中，第(1)项中，所述人类免疫球蛋白IgG各亚型为IgG1、IgG2、IgG3或IgG4。3. The fusion protein according to claim 2, wherein in item (1), each subtype of the human immunoglobulin IgG is IgG1, IgG2, IgG3 or IgG4.4.根据权利要求2所述的融合蛋白，其中，第(1)项中，所述Fc段为IgG4的全长Fc段。4. The fusion protein of claim 2, wherein, in item (1), the Fc segment is a full-length Fc segment of IgG4.5.根据权利要求2所述的融合蛋白，其中，第(3)项中，所述信号肽的氨基酸序列如SEQID NO：9所示。5 . The fusion protein according to claim 2 , wherein, in item (3), the amino acid sequence of the signal peptide is shown in SEQ ID NO: 9. 6 .6.根据权利要求2所述的融合蛋白，其中，第(4)项中，所述Linker的氨基酸序列如SEQID NO：11或SEQ ID NO：13所示。6 . The fusion protein according to claim 2 , wherein, in item (4), the amino acid sequence of the Linker is shown in SEQ ID NO: 11 or SEQ ID NO: 13. 7 .7.根据权利要求1或2所述的融合蛋白，其氨基酸序列如SEQ ID NO：15至SEQ ID NO：18中的任一序列所示。7 . The fusion protein according to claim 1 or 2 , wherein the amino acid sequence is shown in any one of SEQ ID NO: 15 to SEQ ID NO: 18. 8 .8.一种分离的多核苷酸，其编码权利要求1至7中任一权利要求所述的融合蛋白。8. An isolated polynucleotide encoding the fusion protein of any one of claims 1-7.9.根据权利要求8所述的多核苷酸，其核苷酸序列如SEQ ID NO：19至SEQ ID NO：22中的任一序列或其简并性序列所示。9. The polynucleotide of claim 8, wherein the nucleotide sequence is shown in any one of SEQ ID NO: 19 to SEQ ID NO: 22 or a degenerate sequence thereof.10.一种重组载体，其含有权利要求8或9所述的多核苷酸。10. A recombinant vector comprising the polynucleotide of claim 8 or 9.11.根据权利要求10所述的重组载体，其为质粒、重组腺病毒、重组腺相关病毒或噬菌体。11. The recombinant vector of claim 10, which is a plasmid, a recombinant adenovirus, a recombinant adeno-associated virus, or a bacteriophage.12.根据权利要求11所述的重组载体，其中所述质粒为真核表达质粒或原核表达质粒。12. The recombinant vector of claim 11, wherein the plasmid is a eukaryotic expression plasmid or a prokaryotic expression plasmid.13.根据权利要求11所述的重组载体，其中所述质粒为pCDNA3.1、pDC315或pAAV-MCS。13. The recombinant vector of claim 11, wherein the plasmid is pCDNA3.1, pDC315 or pAAV-MCS.14.一种重组宿主细胞，其包含权利要求10至13中任一权利要求所述的重组载体。14. A recombinant host cell comprising the recombinant vector of any one of claims 10-13.15.根据权利要求14所述的重组宿主细胞，其中，所述宿主细胞为细菌。15. The recombinant host cell of claim 14, wherein the host cell is a bacterium.16.根据权利要求15所述的重组宿主细胞，其中，所述宿主细胞为大肠杆菌。16. The recombinant host cell of claim 15, wherein the host cell is Escherichia coli.17.根据权利要求14所述的重组宿主细胞，其中，所述宿主细胞为真菌。17. The recombinant host cell of claim 14, wherein the host cell is a fungus.18.根据权利要求14所述的重组宿主细胞，其中，所述宿主细胞为真核表达宿主细胞。18. The recombinant host cell of claim 14, wherein the host cell is a eukaryotic expression host cell.19.根据权利要求18所述的重组宿主细胞，其中，所述宿主细胞为293细胞。19. The recombinant host cell of claim 18, wherein the host cell is a 293 cell.20.一种组合物，其包含权利要求1至7中任一权利要求所述的融合蛋白或者权利要求8或9所述的多核苷酸或者权利要求10至13中任一权利要求所述的重组载体或者权利要求14至19中任一权利要求所述的重组宿主细胞。20. A composition comprising the fusion protein of any one of claims 1 to 7 or the polynucleotide of claim 8 or 9 or the polynucleotide of any one of claims 10 to 13 A recombinant vector or the recombinant host cell of any one of claims 14-19.21.根据权利要求20所述的组合物，其还包含药学上可接受的载体。21. The composition of claim 20, further comprising a pharmaceutically acceptable carrier.22.根据权利要求20所述的组合物，其还包含药学上可接受的赋形剂。22. The composition of claim 20, further comprising a pharmaceutically acceptable excipient.23.权利要求1至7中任一权利要求所述融合蛋白或者权利要求8或9所述的多核苷酸或者权利要求10至13中任一权利要求所述的重组载体或者权利要求14至19中任一权利要求所述的重组宿主细胞或者权利要求21或22所述的组合物在制备抗肿瘤药物中的用途；其中，所述肿瘤为选自肺癌、肝癌、结肠癌、乳腺癌、肾癌、神经胶质瘤和黑色素瘤中的一种或多种。23. The fusion protein of any one of claims 1 to 7 or the polynucleotide of claim 8 or 9 or the recombinant vector of any one of claims 10 to 13 or claims 14 to 19 Use of the recombinant host cell described in any claim or the composition described in claim 21 or 22 in the preparation of an antitumor drug; wherein the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, breast cancer, kidney cancer One or more of cancer, glioma, and melanoma.24.根据权利要求23所述的用途，其中，所述肝癌为肝癌移植瘤。24. The use according to claim 23, wherein the liver cancer is a liver cancer xenograft.

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