WO2023109835A1 - Vegf-crm197 recombinant fusion protein vaccine, and preparation method therefor and use thereof - Google Patents

Abstract

Provided in the present invention are a VEGF-CRM197 recombinant fusion protein vaccine, and a preparation method therefor and the use thereof. Specifically, provided in the present invention is that truncated VEGF, namely a VEGF1-107 antigen fragment, which loses the biological activity of VEGF but retains its immunogenicity, is fused with diphtheria toxin mutant CRM197 for recombinant expression to form a VEGF recombinant fusion protein. After being used in combination with a liquid adjuvant, the VEGF recombinant fusion protein can induce mice and rhesus monkeys to produce high-titer antibodies and block the binding of VEGF-A to a receptor thereof, thereby inhibiting the promotion effect of VEGF-A on the proliferation of vascular endothelial cells.

Description

A VEGF-CRM197 recombinant fusion protein vaccine and its preparation method and application technical field

The invention belongs to the fields of biotechnology and medicine, and in particular relates to the preparation and application of a vascular endothelial growth factor (VEGF) vaccine.

Background technique

The VEGF/VEGFR signaling pathway is a crucial limiting step in tumor angiogenesis and a key factor in promoting tumor metastasis. Vascular endothelial growth factor A (VEGF-A) is the cytokine most closely related to the execution of tumor angiogenesis and lymphangiogenesis, and activates the VEGF/VEGFR signaling pathway, which can lead to epithelial cell survival, mitosis, metastasis and differentiation, and vascular penetration. It has been confirmed that VEGF-mediated high permeability of blood vessels is closely related to the metastasis of malignant tumors. Studies have shown that tumor tissue hypoxia leads to the gene expression level of VEGF-A, and the expression level of VEGF-A is significantly up-regulated in many tumor tissues, such as non-small cell lung cancer, colorectal cancer, breast cancer, etc. Therefore, inhibiting angiogenesis by blocking the VEGF/VEGFR signaling pathway is a very promising cancer treatment. Humanized monoclonal antibodies to VEGF have been widely used in the treatment of metastatic colorectal cancer and lung cancer, and it has been clinically proven that VEGF monoclonal antibodies can significantly prolong the survival of patients with metastatic colorectal cancer and lung cancer. Although tumor immunotherapy has made major breakthroughs in the fields of immunosuppressants and CAR-T, both immunosuppressant monoclonal antibodies and CAR-T treatments are very expensive. Due to factors such as high dosage and high production cost of monoclonal antibody drugs, long-term use can easily cause severe allergic reactions.

In recent years, with the rapid development and cross penetration of related disciplines such as oncology, immunology, and molecular biology, the use of tumor vaccines that activate the body's specific anti-tumor immune function to treat cancer has become a research hotspot in the field of malignant tumor treatment. Tumor vaccines activate tumor-specific immune responses through tumor-associated antigens, and have achieved the purpose of killing and eliminating tumor cells. It is a therapeutic active immunotherapy method. Cancer vaccine strategies mainly include peptide vaccines, DNA vaccines, and antigen-shocking dendritic cells. So far, a small number of anti-angiogenic vaccines targeting VEGF or VEGFR have been studied in the early stage, and have achieved good growth inhibition effects in preclinical studies. However, conventional peptide vaccines lack sufficient immunogenicity to induce neutralizing antibodies in vivo, and VEGF vaccines containing space epitopes usually contain VEGF biological activity, leading to the introduction of VEGF active components during vaccine injection. Currently reported cancer vaccines targeting angiogenesis mainly adopt two strategies. One of the VEGF vaccines is VEGF121, which contains three mutations of VEGFR2 binding sites R82E, K82E, and H82E, and the biological activity of the mutant VEGF is greatly weakened. However, the results of clinical trials showed that the neutralizing antibody induced by the vaccine was weak and could not effectively block the binding of VEGF165 to its receptor.

Another reported VEGF vaccine is the hVEGF26-104 synthetic polypeptide vaccine, which contains C51A-C60A mutations to ensure that it does not contain VEGF biological activity. Recent reports have shown that although a certain anti-VEGF antibody titer can be observed in cynomolgus monkeys immunized with hVEGF26-104 synthetic peptide vaccine, the peptide vaccine did not induce significant anti-human VEGF165 antibodies and did not cause VEGF concentration levels in phase I clinical trials. decreased, and no clinical benefit was observed. Therefore, the important problem facing the design and preparation of VEGF vaccines is how to break through immune tolerance, stimulate homologous proteins to generate immune responses, and produce neutralizing antibodies against VEGF. On the one hand, peptide vaccines have the defect of low immunogenicity; on the other hand, whether VEGF peptide epitopes can stimulate sufficient anti-VEGF neutralizing antibodies has not yet been clinically confirmed.

Therefore, there is an urgent need in the art to develop a vaccine that has no VEGF biological activity, high immunogenicity, and better effect of induced anti-VEGF antibodies.

Contents of the invention

The object of the present invention is to provide a VEGF fusion protein vaccine with no VEGF biological activity and high immunogenicity, which comprises a recombinant fusion protein in which VEGF antigen fragments (1-107) are fused with diphtheria toxin mutant CRM197. The vaccine does not have VEGF biological activity, but has strong immunogenicity. After being combined with a liquid adjuvant, it can break the immune tolerance of the immune body and induce the body to continuously produce anti-VEGF neutralizing antibodies.

The first aspect of the present invention provides a recombinant fusion protein, the fusion protein has the structure shown in formula I:

Z1-Z2-Z3 (I)

Wherein, Z1 is a VEGF antigen fragment element;

Z2 is a connecting peptide element or none; and

Z3 is a diphtheria toxin mutant CRM197 protein element;

"-" indicates a peptide bond or a peptide linker connecting the above elements;

The VEGF antigen fragment has an amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 6, which loses VEGF biological activity but retains immunogenicity; and the diphtheria toxin mutant CRM197 protein has such as SEQ ID NO : Amino acid sequence shown in 7.

In another preferred example, the VEGF antigen fragment has the amino acid sequence shown in SEQ ID NO:5.

In another preferred example, the VEGF antigen fragment has the amino acid sequence shown in SEQ ID NO:6.

In another preferred example, the length of the peptide linker is 0-15 amino acids, preferably 0-10 amino acids, more preferably 0-5 amino acids.

In another preferred example, the amino acid sequence of the fusion protein has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

In another preferred example, the amino acid sequence of the fusion protein is shown in SEQ ID NO:1.

In another preferred example, the amino acid sequence of the fusion protein is shown in SEQ ID NO:2.

In another preferred embodiment, the amino acid of the fusion protein has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, preferably, at least 95% sequence identity identity, more preferably at least 96%, 97%, 98%, or 99% sequence identity.

In another preferred example, the amino acid sequence of the fusion protein is shown in SEQ ID NO:9.

In another preferred example, the amino acid sequence of the fusion protein is shown in SEQ ID NO:12.

In another preferred example, the fusion protein has the following characteristics:

(1) The VEGF antigen fragment is obtained by truncating the C-terminal 14 amino acid residues on the basis of the amino acid sequence of VEGF121 shown in SEQ ID NO:8;

(2) The VEGF antigen fragment does not have VEGF biological activity;

(3) The VEGF antigen fragment can induce the production of anti-VEGF165 antibody and neutralizing antibody in vivo.

The second aspect of the present invention provides a polynucleotide encoding the recombinant fusion protein as described in the first aspect of the present invention.

In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA sequence, RNA sequence, or a combination thereof.

In another preferred example, the polynucleotide additionally contains auxiliary elements selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), or a combination thereof at the flank of the ORF of the fusion protein.

In another preferred example, the polynucleotide encodes a fusion protein having the structure of formula I:

Z1-Z2-Z3 (I)

Wherein, Z1 is a VEGF antigen fragment;

Z2 is linker peptide or none; and

Z3 is a diphtheria toxin mutant CRM197 protein;

"-" indicates a peptide bond or a peptide linker linking the above elements.

In another preferred example, the VEGF antigen fragment has the amino acid sequence shown in SEQ ID NO:5.

In another preferred example, the VEGF antigen fragment has the amino acid sequence shown in SEQ ID NO:6.

In another preferred example, the diphtheria toxin mutant CRM197 protein has the amino acid sequence shown in SEQ ID NO:7.

In another preferred example, the polynucleotide encodes a recombinant fusion protein as shown in SEQ ID NO:1, which has a nucleotide sequence as shown in SEQ ID NO:3.

In another preferred embodiment, the polynucleotide encodes a recombinant fusion protein as shown in SEQ ID NO:2, which has a nucleotide sequence as shown in SEQ ID NO:4.

In another preferred example, the polynucleotide encodes a recombinant fusion protein as shown in SEQ ID NO: 9, the N-terminal of the fusion protein contains a sumo tag, and it has a nucleoside as shown in SEQ ID NO: 10 acid sequence.

The third aspect of the present invention provides an expression vector containing the polynucleotide sequence described in the second aspect of the present invention.

In another preferred example, the expression vector is used to express the recombinant fusion protein.

In another preferred example, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

In another preferred example, the expression vector is a prokaryotic expression vector.

In another preferred example, the expression vector is a eukaryotic expression vector, which can be applied to construct a eukaryotic cell line expressing the VEGF fusion protein vaccine.

In another preferred example, the vector contains two open reading frames, one of which contains the polynucleotide sequence described in the second aspect of the present invention and the nucleotide sequence encoding the sumo tag, and the core encoding the sumo tag The nucleotide sequence is at the 5' end of the polynucleotide sequence, and the other open reading frame includes the nucleotide sequence encoding Escherichia coli disulfide bond isomerase DsbC.

In another preferred example, the expression vector is an expression vector containing two open reading frames, wherein one open reading frame contains the nucleotide sequence shown in SEQ ID NO: 10, and the other open reading frame contains the coding sequence of large intestine The nucleotide sequence of bacillus disulfide bond isomerase DsbC is shown in SEQ ID NO:11.

The fourth aspect of the present invention provides a host cell containing the expression vector of the third aspect of the present invention or the polynucleotide of the second aspect of the present invention integrated in the genome.

In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is a prokaryotic cell.

In another preferred embodiment, the host cell is Escherichia coli.

In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or combinations thereof.

In another preferred embodiment, the host cell is selected from the group consisting of BL21 (DE3), Rosetta (DE3), and Origami B (DE3).

In another preferred example, the host cell is Chinese hamster ovary cell (CHO).

A fifth aspect of the present invention provides a method for preparing a recombinant fusion protein as described in the first aspect of the present invention, comprising the following steps:

(i) cultivating the host cell described in the fourth aspect of the present invention;

(ii) using an inducer to induce the host cell to express the recombinant fusion protein, thereby obtaining the expressed recombinant fusion protein;

(iii) isolating the expressed recombinant fusion protein, thereby obtaining the isolated recombinant fusion protein.

In another preferred example, in the host cell, the expressed recombinant fusion protein is expressed in the form of inclusion bodies of the recombinant fusion protein.

In another preferred example, the separation in step (iii) includes denaturation and renaturation of inclusion bodies of the recombinant fusion protein.

In another preferred example, the method for denaturation and renaturation of the inclusion body of the recombinant fusion protein comprises the following steps:

(i) washing the inclusion body of the recombinant fusion protein with a washing buffer;

(ii) mix the recombinant fusion protein inclusion body with process water not exceeding the mass volume of the recombinant fusion protein inclusion body, and then dissolve the recombinant fusion protein inclusion body with a denaturing solution to obtain a dissolved recombinant fusion protein ;

(iii) Refolding the dissolved recombinant fusion protein with a refolding solution at a refolding temperature of 16-25° C. to obtain a refolded recombinant fusion protein.

In another preferred example, the preparation method of the recombinant fusion protein comprises the following steps:

(1) Cultivate the BL21(DE3) strain host cell as described in the fourth aspect of the present invention until the OD600 value is 10-20, and add an inducer to obtain the BL21(DE3) strain containing the inclusion body of the recombinant VEGF fusion protein;

(2) Treat the BL21 (DE3) strain containing the recombinant VEGF fusion protein inclusion body with a bacteriostasis buffer, collect the recombinant VEGF fusion protein inclusion body, and wash the inclusion body with a bacteriostasis buffer, denature, refold and Purify to isolate the recombinant VEGF fusion protein.

In another preferred embodiment, the medium described in step (1) is a fully synthetic medium;

The fully synthetic medium contains 1-4g/L KH ₂ PO ₄ , 1-5g/L K ₂ HPO ₄ ·3H ₂ O, 2-10g/L (NH ₄ ) ₂ SO ₄ , 0.1-2g/L MgSO ₄ · 7H ₂ O, 10-50wt% glucose, and supplemented with 10-5-wt% glycerol and 10-200g/L ammonium sulfate during the cultivation process.

In another preferred embodiment, the bacteriostasis buffer contains 0.4-2mol/L urea, 0.1-1.0% Triton X-100, and 0.1-1.0% Triton X-114.

In another preferred example, the number of washings in step (2) is 2-5 times.

In another preferred example, the inclusion body is first mixed with process water not exceeding the mass volume of the inclusion body, and then dissolved with the following denaturing solution:

In another preferred example, the denaturation solution contains 6 mol/L guanidine hydrochloride and 1-50 mM DTT.

In another preferred example, the denaturation solution is firstly diluted with 8 mol/L urea at a ratio of 1:4, and then refolded by diluting in the refolding solution.

In another preferred example, the refolding liquid contains 0.1%-0.5% PEG4000.

In another preferred example, the annealing temperature is 16°C-25°C.

In another preferred embodiment, the purification is performed using anion exchange chromatography, and the anion exchange medium used is Q sepharose.

In another preferred example, the recombinant fusion protein is expressed in a soluble form by adding a tag or co-expressing with a partner molecule that assists in protein folding.

In another preferred example, the preparation method of the recombinant fusion protein comprises the following steps:

(1) cultivating the host cell described in the fourth aspect of the present invention, using an inducer to induce the host cell to express the recombinant fusion protein, thereby obtaining the expressed recombinant fusion protein;

(2) Collect the expression supernatant after the bacterial cell is crushed, and make it rough and pure by affinity chromatography;

(3) Use specific protease to remove the tag, and then use affinity chromatography to remove the tag and added protease;

(4) Obtain high-purity recombinant fusion protein through anion exchange chromatography and/or molecular sieve chromatography purification.

In another preferred example, the preparation method of the recombinant fusion protein comprises the following steps:

(1) Apply the eukaryotic expression vector provided by the third aspect of the present invention to construct a Chinese hamster ovary cell (CHO) cell line expressing recombinant VEGF fusion protein;

(2) High-density fermentation culture, such as the CHO cells mentioned above, is fermented and cultured at a fermentation temperature of 30-38°C for 7-21 days until the cell density is 10×10 ⁶ -20×10 ⁶ /ml;

(3) collecting the fermentation supernatant of step (2), and purifying by hydrophobic chromatography, anion exchange chromatography and cation exchange chromatography respectively.

The sixth aspect of the present invention provides a pharmaceutical composition comprising the recombinant fusion protein described in the first aspect of the present invention and a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutically acceptable carrier contains liquid, preferably water, saline or buffer.

In another preferred example, the carrier also contains auxiliary substances, preferably fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances and the like.

In another preferred example, the vector also contains a cell transfection reagent.

In another preferred embodiment, the composition is a vaccine composition.

In another preferred embodiment, the vaccine composition comprises the recombinant fusion protein described in the first aspect of the present invention and a vaccine acceptable carrier, and the vaccine acceptable carrier is preferably a pharmaceutically acceptable carrier.

In another preferred example, the vaccine composition may be a dual vaccine or a multiple vaccine.

In another preferred example, the vaccine composition further contains an adjuvant.

In another preferred example, the adjuvant includes: granular and non-granular adjuvants.

In another preferred example, the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or combinations thereof;

In another preferred example, the non-granular adjuvant is selected from the group consisting of muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as Mycobacterium (Mycobacterium tuberculosis, BCG), Brevibacterium, Bacillus pertussis, propolis, or combinations thereof.

In another preferred embodiment, the adjuvant is selected from the group consisting of Montanide ISA 51 VG, aluminum phosphate adjuvant, MF59, AS04, or a combination thereof.

In another preferred example, the amount of VEGF recombinant fusion protein in each dose of the vaccine composition is 0.1-5 mg.

In another preferred example, the vaccine composition is in the form of injection.

The seventh aspect of the present invention provides a recombinant fusion protein as described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, and the polynucleotide described in the first aspect of the present invention Use of the host cell according to the fourth aspect, and/or the pharmaceutical composition according to the sixth aspect of the present invention in the preparation of medicines for treating and/or preventing diseases.

In another preferred embodiment, the disease is selected from the group consisting of tumor (or cancer), macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, diabetic macular edema, or a combination thereof.

In another preferred example, the tumor (or cancer) includes a solid tumor.

In another preferred example, the solid tumor is selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer cancer, ovarian cancer, nasopharyngeal cancer, oral cavity cancer, osteosarcoma, glioma, bladder cancer, or a combination thereof.

The eighth aspect of the present invention provides a method for treating and/or preventing diseases, the method comprising administering an effective amount of the pharmaceutical composition as described in the sixth aspect of the present invention to a subject in need.

In another preferred embodiment, the disease is selected from the group consisting of tumor (or cancer), macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, diabetic macular edema, or a combination thereof.

In another preferred example, the tumor (or cancer) includes a solid tumor.

In another preferred example, the solid tumor is selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer cancer, ovarian cancer, nasopharyngeal cancer, oral cavity cancer, osteosarcoma, glioma, bladder cancer, or a combination thereof.

The ninth aspect of the present invention provides a method for immunizing VEGF recombinant fusion protein vaccine, comprising the steps of:

(i) emulsifying after mixing the VEGF recombinant fusion protein described in the first aspect of the present invention with an adjuvant to obtain an emulsified VEGF recombinant fusion protein vaccine;

(ii) Vaccine the subject to be vaccinated with the emulsified VEGF recombinant fusion protein vaccine.

In another preferred example, the adjuvant is a liquid adjuvant.

In another preference, the liquid adjuvant is Montanide ISA 51 VG;

In another preferred example, the "emulsification after mixing the VEGF recombinant fusion protein with the adjuvant" is specifically passing the VEGF recombinant fusion protein and Montanide ISA 51 VG at a volume ratio of 1: (0.5-2) through two syringes. Joint connection mixing, first push back and forth slowly 10-30 times, then push back and forth quickly 30-60 times;

In another preferred example, the vaccination method is as follows: immunization once a week at a dose of 0.05-2 mg/kg, 4 times in total.

In another preferred example, the liquid adjuvant is an aluminum phosphate adjuvant, and the recombinant VEGF fusion protein and the aluminum phosphate adjuvant are mixed and emulsified at a volume ratio of 1:(0.2-5).

In another preferred example, the liquid adjuvant is MF59, and the recombinant VEGF fusion protein and MF59 adjuvant are mixed and emulsified at a volume ratio of 1:(0.2-5).

In another preferred example, the liquid adjuvant is AS04, and the recombinant VEGF fusion protein and the AS04 adjuvant are mixed and emulsified at a volume ratio of 1:(0.2-5).

In another preferred example, the subject to be vaccinated is a human or a non-human mammal.

In another preferred example, the non-human mammal is selected from the group consisting of mice, rats, rabbits, and rhesus monkeys.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the results of detection of VEGF biological activity of VEGF fragments with different lengths.

Figure 2 shows the results of immunogenicity testing of VEGF fragments with different lengths.

Fig. 3 shows the whole bacterial protein map of the recombinant expression of the recombinant fusion protein vaccine.

Figure 4 shows a schematic diagram of the SDS-PAGE purity of the recombinant fusion protein vaccine after preparation.

Figure 5 shows a schematic diagram of the RP-HPLC purity of the recombinant fusion protein vaccine after preparation.

Figure 6 shows the results of detection of serum antibody titers in mice immunized with recombinant fusion protein vaccine

Figure 7 shows the detection results of neutralizing antibodies in serum of mice immunized with recombinant fusion protein vaccine.

Figure 8 shows the detection results of inhibition of VEGF-stimulated proliferation of vascular endothelial cells by serum of mice immunized with recombinant fusion protein vaccine.

Figure 9 shows the results of detection of serum antibody titers of recombinant fusion protein vaccine (sequence such as SEQ ID NO: 2) immunized mice.

Fig. 10 has shown the detection result of serum antibody titer of recombinant fusion protein vaccine (sequence such as SEQ ID NO: 12) immunized mice.

Figure 11 shows the result of the sequence alignment of recombinant fusion protein vaccine sequences SEQ ID NO:1 and SEQ ID NO:2.

Figure 12 shows the results of antibody titer detection in rhesus macaques immunized with the recombinant fusion protein vaccine.

Figure 13 shows the detection results of the neutralizing antibody of the rhesus macaque antibody immunized with the recombinant fusion protein vaccine.

Figure 14 shows the results that the antibody significantly inhibits tumor growth after immunization with the recombinant fusion protein vaccine, in which C021 is the recombinant fusion protein vaccine (comprising the sequence shown in SEQ ID NO: 1).

Figure 15 shows that the antibody significantly prolongs the survival of tumor-bearing mice after immunization with the recombinant fusion protein vaccine.

Figure 16 shows the comparison results of antibody titer detection after VEGF121-CRM197 and VEGF107-CRM197 immunized mice.

Figure 17 shows the sequence alignment results of the recombinant fusion protein whose sequences are shown in SEQ ID NO: 2 and 12.

Detailed ways

After extensive and in-depth research, the inventors discovered for the first time that synthetic polypeptides of VEGF cannot stimulate high antibody titers in animals, while the truncated VEGF1-107 fragment of VEGF121 loses biological activity of VEGF but retains immunogenicity, and can Stimulate the production of high-titer antibodies in animals. The recombinant fusion protein prepared by fusing the VEGF antigen fragment (1-107) with the diphtheria toxin mutant CRM197 has strong immunogenicity, and can break the immune tolerance of the immune body after being combined with a liquid adjuvant, and induce the body to continuously produce anti-VEGF neutralization Antibody. Experiments prove that, compared with VEGF synthetic polypeptides, the VEGF recombinant fusion protein of the present invention has stronger affinity with receptor protein kinase domain receptors, and the antibody titer produced in animals after immunization is higher. On this basis, the present invention has been accomplished.

the term

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

VEGF1-107

As used in the present invention, the terms "VEGF1-107 fragment", "VEGF antigen fragment (1-107)" and "VEGF107" are used interchangeably.

The VEGF1-107 fragment of the present invention is based on the VEGF121 amino acid sequence shown in SEQ ID NO: 8, truncated 14 amino acid residues at the C-terminus, and retains the 107th amino acid residue from the N-terminal to the C-terminal of the VEGF antigen fragment . The obtained shorter VEGF1-107 fragment loses the biological activity of VEGF but retains the immunogenicity, minimizes the safety risk caused by the biological activity of VEGF, improves the immune efficacy of the vaccine, and can reduce the incidence of non-specific antibodies to a certain extent. produce.

VEGF121 is a secreted vascular endothelial growth factor, which is the smallest molecular weight VEGF splice body naturally present in the human body. This molecule can stimulate VEGF receptors and induce the growth of vascular endothelial cells through signal transduction.

In another preferred embodiment, the VEGF1-107 fragment of the present invention has the amino acid sequence shown in SEQ ID NO:5.

In another preferred embodiment, the VEGF1-107 fragment of the present invention further comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:5, for example, the VEGF1-107 fragment It has an amino acid sequence as shown in SEQ ID NO: 6, which has three amino acid mutations compared to SEQ ID NO: 5, specifically, Arg at position 82 is mutated to Glu, Lys at position 84 is mutated to Glu, and Lys at position 84 is mutated to Glu. His at position 86 is mutated to Glu.

The VEGF1-107 fragment of the present invention has the following amino acid sequence:

CRM197

As used in the present invention, the term "CRM197" refers to the diphtheria toxin mutant CRM197, specifically, the glycine at the 52nd position of the diphtheria toxin is mutated to glutamic acid, which has the amino acid sequence shown in SEQ ID NO:7. The mutant toxin A fragment cannot combine with elongation factor II in the nucleus, making it lose the cytotoxic effect, but the antigenicity and immunogenicity are still basically consistent with the natural diphtheria toxin.

fusion protein

As used in the present invention, the terms "recombinant VEGF fusion protein", "VEGF recombinant fusion protein" and "VEGF fusion protein", "recombinant fusion protein of the present invention" can be used interchangeably, all referring to VEGF antigen fragment (1-107) ( VEGF1-107 fragment) and the recombinant fusion protein VEGF107-CRM197 obtained by fusion of diphtheria toxin mutant CRM197.

In another preferred example, the structure of the fusion protein is shown as Z1-Z2-Z3 (Formula I),

Wherein, Z1 is a VEGF antigen fragment element; Z2 is a connecting peptide element or none; and Z3 is a diphtheria toxin mutant CRM197 protein element; "-" indicates a peptide bond or a peptide linker connecting the above elements.

In another preferred example, the coding sequence of the fusion protein is shown in SEQ ID NO:1 or SEQ ID NO:2.

As used in the present invention, the term "fusion protein" also includes variant forms of the fusion protein (such as the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2) having the above-mentioned activity. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions or substitutions at the C-terminal and/or N-terminal One or several 15 (usually within 3, preferably within 2, more preferably within 1) amino acids are deleted. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein. Furthermore, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (eg, cyclic peptides).

The present invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used in the present invention, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the function or activity of the fusion protein of the present invention. The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused to a leader sequence, a secretory sequence, or a tag sequence such as 6×His). According to the teaching of the present invention, these fragments, derivatives and analogs belong to the range known to those skilled in the art.

One class of preferred active derivatives refers to that compared with the amino acid sequence of formula I, at most 3, preferably at most 2, more preferably at most 1 amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A

initial residue	representative replacement	preferred substitution
Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
Asn(N)	Gln; His; Lys; Arg	Gln
Asp(D)	Glu	Glu
Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
Glu(E)	Asp	Asp
Gly(G)	Pro;	Ala
His(H)	Asn; Gln; Lys; Arg	Arg
Ile (I)	Leu; Val; Met; Ala; Phe	Leu
Leu(L)	Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
Met(M)	Leu; Phe; Ile	Leu
Phe(F)	Leu; Val; Ile; Ala; Tyr	Leu
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val(V)	Ile; Leu; Met; Phe;	Leu

The invention also provides analogs of the fusion proteins of the invention. The difference between these analogs and the polypeptide shown in any one of SEQ ID NO.: 1-2 may be a difference in amino acid sequence, or a modification that does not affect the sequence, or both. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during synthesis and processing or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

Expression Vectors and Host Cells

The present invention also relates to a vector comprising a polynucleotide encoding the fusion protein of the present invention, and a host cell produced by genetic engineering with the vector of the present invention or the coding sequence of the fusion protein of the present invention, and producing the fusion protein of the present invention through recombinant techniques Methods.

The polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the fusion protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

In the present invention, the polynucleotide sequence encoding the fusion protein can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct an expression vector containing the fusion protein coding DNA sequence of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell can be a prokaryotic cell (such as Escherichia coli), or a lower eukaryotic cell, or a higher eukaryotic cell, such as yeast cells or mammalian cells (including human and non-human mammals). Representative examples include: Escherichia coli, insect cells, SF9, Hela, HEK293, CHO, yeast cells, etc. In a preferred embodiment of the present invention, Escherichia coli (such as BL21(DE3), Rosetta(DE3), JM109, etc.) is selected as the host cell. In another preferred embodiment of the present invention, CHO cells are selected as host cells.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide or fusion protein encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

peptide linker

The invention provides a fusion protein which optionally contains a peptide linker. Peptide linker size and complexity may affect protein activity. In general, the peptide linker should be of sufficient length and flexibility to ensure that the two proteins being linked have sufficient degrees of freedom in space to function. In a preferred embodiment of the present invention, the length of the peptide linker is generally 0-15 amino acids, preferably 0-10 amino acids, more preferably 0-5 amino acids.

pharmaceutical composition

The invention also provides a pharmaceutical composition. The pharmaceutical composition contains the above-mentioned fusion protein, and a pharmaceutically acceptable carrier, diluent, stabilizer and/or thickener, and can be prepared as lyophilized powder, tablet, capsule, syrup, solution or suspension Liquid agent type.

"Pharmaceutically acceptable carrier or excipient" means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and must be of sufficient purity and sufficient Low toxicity. "Compatibility" here means that each component in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

Compositions may be liquid or solid, such as powders, gels or pastes. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as

), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

The compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration. The pharmaceutical composition is used for (a) treating or preventing cancer or tumors (especially solid tumors); (b) treating or preventing retinal vein occlusion secondary macular edema, wet age-related macular degeneration and diabetic Macular edema and other diseases; (c) Inducing the production of neutralizing antibodies that block the binding of VEGF to receptors.

The solid tumor is selected from the group consisting of lung cancer, non-small cell lung cancer, colorectal cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, melanoma, kidney cancer, prostate cancer, cervical cancer, ovarian cancer, nasal cancer, Pharyngeal cancer, oral cavity cancer, osteosarcoma, glioma, bladder cancer, or a combination thereof.

In addition, the pharmaceutical composition can be administered to a subject in need alone or in combination with other pharmaceutical preparations for the treatment or prevention of the disease.

vaccine composition

The pharmaceutical composition provided by the invention is preferably a vaccine composition. The vaccine composition comprises the recombinant fusion protein described in the first aspect of the present invention and a vaccine acceptable carrier, preferably a pharmaceutically acceptable carrier.

In another preferred example, the vaccine composition further contains an adjuvant, and the adjuvant is preferably a liquid adjuvant. After the vaccine composition of the present invention is mixed and emulsified with a liquid adjuvant, it can be inoculated in humans or non-human mammals to induce the production of anti-VEGF neutralizing antibodies in vivo.

The present invention also provides a method for immunizing with VEGF recombinant fusion protein vaccine, comprising the steps of:

(i) emulsifying after mixing the VEGF recombinant fusion protein with the adjuvant to obtain the emulsified VEGF recombinant fusion protein vaccine;

(ii) Vaccine the subject to be vaccinated with the emulsified VEGF recombinant fusion protein vaccine.

In another preferred embodiment, the adjuvant is selected from the group consisting of Montanide ISA 51 VG, aluminum phosphate adjuvant, MF59, AS04, or a combination thereof.

Compared with prior art strategies, the present invention mainly has the following advantages:

(1) The present invention selects VEGF1-107 fragments that do not have VEGF biological activity but have strong immunogenicity as antigens, which are more likely to induce in vivo neutralizing antibodies that block the binding of VEGF to receptors;

(2) The present invention provides a recombinant VEGF fusion protein vaccine expressed by fusion of human VEGF1-107 fragments and diphtheria toxin mutant CRM197, wherein CRM197 can significantly improve the immunogenicity of the vaccine antigen;

(3) The present invention provides a variety of preparation methods of the VEGF fusion protein vaccine of the present invention;

(4) Emulsifying the recombinant VEGF fusion protein vaccine of the present invention with a liquid adjuvant can further improve the immunogenicity of the vaccine antigen.

The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1: Screening for VEGF antigen fragments with low VEGF biological activity and high immunogenicity

VEGF121(1-121), VEGF107(1-107) and VEGF82(24-105) were prepared by Escherichia coli expression system respectively, and three VEGF fragments were detected by VEGF-responsive luciferase reporter cell line after preparation.

The VEGF-responsive cell lines were plated in a 96-well plate. After the cells were fixed, three VEGF fragments were added respectively. The VEGF fragments were gradually diluted from 500ng/ml. After incubation for 24 hours, a luciferase substrate was added to detect the luminescence value.

As shown in Figure 1, higher concentrations of VEGF121 can induce luciferase expression through signaling pathways by binding to VEGFR2 on the cell membrane of the responding cell line, and the concentration and fluorescence values show an S-shaped curve; Luciferase expression was not induced, indicating that VEGF107 and VEGF82 do not have VEGF biological activity.

The mice were immunized with VEGF121, VEGF107 and VEGF82 with complete Freund's adjuvant, 10 μg each time, 8 mice in each group, 4 times in a week, 1 week after the second immunization and 1 week after the fourth immunization Blood was collected separately, and the antibody titer was detected with VEGF165.

The results are shown in Figure 2. One week after the second immunization, 100% of the V107 group became positive, only one of the V121 and V82 groups became positive, and none of the V33 group became positive. One week after the four immunizations, the antibody titer in the V107 group was the highest, while there were no transpositive mice in the V82 group. This result indicated that VEGF107 had the strongest immunogenicity.

Through screening, it is found that VEGF107 (containing 1-107 amino acid sequence) has no biological activity, but has strong immunogenicity, and the VEGF fragment is very suitable for the preparation of VEGF vaccine.

Example 2: Construction and expression of pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC plasmid

The DNA coding sequence of sumoVEGF107-CRM197 (as shown in SEQ ID NO: 10) was synthesized and constructed into the first open reading frame of the pCDFDuet-1 expression plasmid by means of gene synthesis, wherein the amino acid sequence encoding sumo is located at The N-terminus of VEGF107-CRM197 (SEQ ID NO: 1), the amino acid sequence of sumoVEGF107-CRM197 is shown in SEQ ID NO: 9; on the basis of the previous step, the DNA encoding Escherichia coli disulfide bond isomerase DsbC The sequence (shown as SEQ ID NO: 11) was synthesized using gene synthesis and constructed into the second open reading frame of the pCDFDuet-1 expression plasmid. After the identification, the construction of pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC was completed.

Take 2 μL of the constructed pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC plasmid and add it to BL21(DE3) expression engineering bacteria, incubate on ice for 30 minutes, heat shock at 42°C for 90 seconds, incubate on ice for 5 minutes, and then add 500 μL of antibiotic-free LB medium was cultured on a shaker at 37°C for 30 minutes, 50 μL was spread on LB solid medium containing 100 μg/ml streptomycin, and cultured at 37°C overnight.

Pick the single clone on the plate transformed with pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC plasmid and put it in LB liquid medium, cultivate it at 37°C, when the OD600 of the medium reaches 10-20, slowly add glycerol and sulfuric acid Ammonium, adding IPTG with a final concentration of 1mM, and collecting the expression cells after induction at 25°C for 8 hours, and using the SDS-PAGE method to identify the expression status, as shown in Figure 3, sumoVEGF107-CRM197 was expressed in the supernatant fraction.

Embodiment 3: Preparation of recombinant fusion protein VEGF107-CRM197

Get the expressed thalline in Example 2, after the thalline is broken, the supernatant is immediately chromatographed with a Ni affinity column, the washing condition is 10% Buffer B (containing about 50mM imidazole), and the elution condition is 50% Buffer B ( Contains about 250mM imidazole). After the eluted product of affinity chromatography was cleaved by sumo tag-specific protease Ulp1, Ni sepharose FF affinity chromatography was used again to collect the flow-through fraction, and the sumo tag still with His tag and the uncleaved tag were removed. Target protein, partial high-affinity label with Ni column.

Concentrate the VEGF107-CRM197 flow-through fraction without the tag after digestion, and continue to use Sephacryl S200 molecular sieve chromatography to refine and purify the target protein peak. The SDS-PAGE identification diagram of the protein sample is shown in Figure 4, and the RP-HPLC analysis As shown in Figure 5, the analytical purity of both is greater than 95%. That is, the recombinant fusion protein vaccine VEGF107-CRM197 was obtained.

Example 4: Emulsification of recombinant fusion protein VEGF107-CRM197 and liquid adjuvant Montanide ISA 51 VG and immunization of mice

Take the recombinant fusion protein VEGF107-CRM197 prepared in Example 3 and dilute it to 0.2mg/ml, take 0.5ml of the diluted fusion protein to a 2ml syringe, and another 0.5ml liquid adjuvant Montanide ISA 51 VG to another In a 2ml syringe, connect the two syringes with a joint, push back and forth slowly for 15 rounds, and then push back and forth as fast as possible for 30 times to complete the emulsification. After emulsification is complete, push all the emulsion into the syringe on one side.

The mice were divided into a test group and a control group, with 8 mice in each group, weighing more than 18 g. The mice in the test group were subcutaneously injected with 100 μL/mouse of the emulsified emulsion, and the mice in the control group were subcutaneously injected with VEGF antigen, immunized once a week, and blood was collected one week after the fourth immunization to determine the titer of anti-VEGF165 antibody and anti-VEGF165 neutralizing antibody .

4.1 Anti-VEGF antibody titer detection in mouse serum after recombinant fusion protein vaccine (containing the sequence shown in SEQ ID NO: 1) immunization:

(1) Coating: Take a 96-well plate for ELISA detection, dilute VEGF165 protein with Na ₂ CO ₃ -NaHCO ₃ , pH 9.6 coating buffer to 0.5 μg/ml, take 100 μL of the diluted VEGF165 protein and add it to 96 Incubate overnight at 2-8°C in each well of the well plate.

(2) Blocking: the next day, the coating solution was discarded, and 150 μL of PBS blocking solution containing 0.05% (v/v) Tween-20 and 1% BSA was added to each well, and incubated at 37°C for 1 hour;

(3) Washing: After discarding the blocking solution, each well was repeatedly washed 3 times with 200 μL of PBS containing 0.05% (v/v) Tween-20, and the liquid in the well was blotted dry;

(4) Dilute the collected mouse serum, take 100 μL of the diluted serum and add it to the leftmost well of the 96-well plate, each mouse is 1 row, doubling dilution from left to right, a total of 12 gradients are diluted, and Using the serum of non-immunized mice as a control, after adding serum, incubate at 37°C for 1 hour; after incubation, discard the incubation solution, and wash repeatedly 3 times as above;

(5) Dilute the secondary antibody HRP enzyme-labeled anti-mouse antibody to 1:5000 with blocking solution, add 100 μL of HRP enzyme-labeled antibody to each well, incubate at 37°C for 1 hour; discard the incubation solution, and wash 5 times repeatedly as above. ;

(6) Color development: add 100 μl TMB to each well, and incubate at room temperature in the dark for 15 minutes;

(7) Termination: add 100 μl 0.5mol/L sulfuric acid to each well to terminate the color development;

(8) After termination, detect the light absorption value of OD450 of each well with a microplate spectrophotometer. Measure the light absorption value at 450nm, and determine the highest dilution factor where the serum sample OD value/saline control group sample OD value ≥ 2.1.

As shown in Figure 6, the geometric mean of the highest dilution factor of 8 mice was calculated as the antibody titer, and the antibody titer was 3×10 ⁶ , which was 100 times higher than that of the VEGF alone antigen group.

4.2 Detection of anti-VEGF neutralizing antibody in serum of immunized mice after recombinant fusion protein vaccine immunization:

Take the 96-well plate for ELISA detection, dilute the VEGF165 protein with Na ₂ CO ₃ -NaHCO ₃ , pH 9.6 coating buffer to 80ng/ml, take 100 μL of the diluted VEGF165 protein and add it to each well of the 96-well plate, Incubate overnight at 2-8°C. The next day, discard the coating solution, add 150 μL of PBS blocking solution containing 0.05% (v/v) Tween-20 and 1% BSA to each well, and incubate at 37°C for 1 hour; washing: after discarding the blocking solution, use 200 μL of PBS containing 0.05% (v/v) Tween-20 was repeatedly washed 3 times, and the liquid in the well was blotted dry; the collected mouse serum was diluted with an initial dilution factor of 3 times, and 100 μL of the diluted serum was added to 96 wells In the leftmost well of the plate, each mouse is 1 row, and the blank group mouse (non-immunized mouse) serum is used as the control, and the dilution ratio is doubled from left to right, and a total of 12 gradients are diluted. After adding the diluted serum Incubate at 37°C for 1 hour; after incubation, discard the incubation solution, and wash repeatedly 3 times as described above; take VEGF receptor VEGFR2 kinase domain receptor (KDR) and Fc fusion protein, dilute to 160ng/ml with blocking solution , and add 100 μL to each well, incubate and block at 37°C for 1 hour; discard the incubation solution, and wash three times as described above; dilute HRP enzyme-labeled anti-VEGFR2 antibody to 1:5000 with blocking solution, add HRP enzyme-labeled antibody 100μL, 37°C, incubate for 1h; discard the incubation solution, and wash 5 times as above; color development: add 100μl TMB to each well, incubate at room temperature for 15min in the dark; termination: add 100μl 0.5mol to each well /L sulfuric acid; after termination, use a microplate spectrophotometer to detect the light absorption value of OD450 of each well.

The results are shown in Figure 7. The higher the OD450 value, the higher the binding content of VEGFR2 kinase domain receptor to VEGF165. In the color development results, in the control serum group, different concentrations of serum did not affect the color development results, while in the recombinant fusion In the protein vaccine immunization group, after incubation of low-dilution serum with VEGF-coated well plates, the binding of VEGFR2 kinase domain receptors to VEGF165 was significantly inhibited, reflecting strong neutralizing antibody activity.

4.3 Detection of inhibition of proliferation of vascular endothelial cells:

experiment material:

Complete medium 1: ECM medium supplemented with 5% FBS (V/V), 1% ECGS and 1% P/S. Store in glass or plastic bottles at 4°C, and the service life shall not exceed the product indication and expiration date.

Complete medium 2: 0.5% FBS (V/V) and 1% P/S were added to the ECM medium. Store in glass or plastic bottles at 4°C, and the service life shall not exceed the product indication and expiration date.

Complete medium 3: Add 10% CCK-8 to complete medium 2 (prepared as needed).

experiment procedure:

(1) Plating: count the cells after digesting the cells, dilute the cells to 3×10 ⁴ cells/ml with complete medium 1, and inoculate them in a 96-well cell culture plate, 100 μl per well. Cultivate for 18-24 hours at 37°C and 5% CO ₂ .

(2) The medium in the 96-well cell culture plate was replaced with complete medium 2, and the cells were starved for 16-24h.

(3) Sample preparation: ①Blank control group: complete medium 2 without VEGF165, 120 μl per well of a 96-well plate, 20 wells in total. ② Control group: The serum of mice in the blank group was diluted 2-fold with complete medium 2 containing 6.25ng/ml VEGF165, and there were 10 gradient concentrations in total. Two wells were made for each gradient, and the final volume of each well was 120μl. ③ Positive control group: avastin (commercialized VEGF monoclonal antibody) was diluted 2-fold with complete medium 2 containing 6.25ng/ml VEGF165, and the final concentration in the first well was 800ng/ml. There were 10 concentration gradients in total, each gradient Make 2 wells with a final volume of 120 μl in each well. ④ Test sample group: The serum of the recombinant fusion protein vaccine immunized group was diluted 2-fold with complete medium 2 containing 6.25ng/ml VEGF165, a total of 10 gradient concentrations, 2 wells for each gradient, and the final volume of each well was 120μl.

All samples to be tested were co-incubated for 2 hours at 37°C and 5% CO ₂ .

(4) Sample addition and co-incubation: take the cell culture plate prepared in step (2), discard the supernatant of each well, add 100 μl of the samples to be tested with different concentration gradients incubated in step (3), and the total volume of each well is 100 μl. Cultivate for 72 hours at 37°C and 5% CO ₂ .

(5) Detection: Add 10 μl of CCK-8 to each well incubated in step (4) for 72 hours, incubate in a 37° C. incubator for 5 hours, and measure the absorbance at 450 nm with a microplate reader.

The test results are shown in Figure 8. Similar to the avastin monoclonal antibody, the recombinant fusion protein vaccine immunization group can significantly inhibit the proliferation of vascular endothelial cells, while the serum of the control group has no obvious inhibition.

Example 5: Recombinant fusion protein (comprising the sequence shown in SEQ ID NO: 2) emulsified with liquid adjuvant Montanide ISA 51 VG and immunized mice

The design, expression and preparation process of the recombinant fusion protein (as shown in SEQ ID NO: 2) are as described in Examples 2 and 3, the difference is that the DNA coding sequence of VEGF107-CRM197 (as shown in SEQ ID NO: 4) The whole sequence was synthesized by means of gene synthesis and constructed on the pCDFDuet-1-DsbC expression plasmid. The sequence alignment results of SEQ ID NO:1 and SEQ ID NO:2 are shown in Figure 11.

The emulsification method and antibody titer detection method are as described in Example 4, the difference is that the blood collection point is 2 weeks after the second immunization.

The test results are shown in Figure 9, the recombinant fusion protein comprising the amino acid sequence of SEQ ID NO: 2 can also induce high-titer anti-VEGF165 antibody titers in mice.

Example 6: Recombinant fusion protein (comprising the sequence shown in SEQ ID NO: 12) emulsified with liquid adjuvant Montanide ISA 51 VG and immunized mice

The design, expression and preparation process of the recombinant fusion protein (shown as SEQ ID NO: 12) are as described in Examples 2 and 3. The sequence alignment result of SEQ ID NO:12 and SEQ ID NO:2 is shown in Figure 17.

The emulsification method and antibody titer detection method are as described in Example 4.

The test results are shown in Figure 10, the recombinant fusion protein comprising the amino acid sequence of SEQ ID NO: 12 can also induce mice to produce high titers of anti-VEGF165 antibody titers.

Embodiment 7: Recombinant fusion protein vaccine immunization rhesus macaque

As described in Example 4, the recombinant VEGF fusion protein (as shown in SEQ ID NO: 1) was emulsified with the adjuvant Montanide ISA 51 VG, and after emulsification, 0.8ml of the emulsion was injected subcutaneously into the biceps brachii of rhesus monkeys. Injection once a week, a total of 4 injections, blood collection 1 week after the fourth injection, anti-VEGF165 antibody titer detection, anti-VEGF165 neutralizing antibody detection and inhibition of vascular endothelial cell proliferation detection.

The monkey serum anti-VEGF165 antibody titer detection method is similar to the anti-VEGF165 antibody titer in Example 4, the only difference is that the secondary antibody is replaced by HRP enzyme-labeled anti-monkey antibody.

The test results of anti-VEGF165 antibody titer in monkey serum are shown in Figure 12. The result shows that the antibody titer exceeds 10 ⁶ , which has significantly exceeded the reported antibody titer of VEGF vaccine in monkeys (not higher than 8000).

The detection methods of anti-VEGF165 neutralizing antibody and inhibition of proliferation of vascular endothelial cells are similar to those described in Example 4. The results are shown in Figure 13. The results show that the recombinant VEGF fusion protein vaccine can stimulate monkeys to produce antibodies that inhibit the binding of VEGF165 to its receptor and inhibit the proliferation of vascular endothelial cells, that is, break immune tolerance, induce the production of anti-VEGF antibodies, and inhibit the production of vascular endothelial cells. Proliferation, thereby inhibiting angiogenesis, and inhibiting tumor growth.

Example 8: Purified antibody inhibits tumor growth after immunization with recombinant fusion protein vaccine

The recombinant VEGF fusion protein was prepared as described in Examples 2 and 3. After preparation, rabbits were immunized with adjuvant. Each rabbit was immunized with 1 mg each time, and immunized four times. Rabbit serum was collected in January after the four times of immunization, and the vaccine was mixed with adjuvant. The agent combination method is as described in Example 4.

Rabbit serum antibody preparation after immunization: Rabbit serum was centrifuged, ammonium sulfate precipitated, collected and refused to obtain the crude antibody extract, and the protein A filler was used to capture the antibody, and the purified antibody was collected by elution with pH 3.0 citric acid buffer .

Rhabdomyosarcoma A673 cells were selected and cultured at 37°C in an environment with a CO2 volume ratio of 5%, and the composition of the growth medium was 90% DMEM+10% FBS. Tumor-bearing link: first wash the cells with PBS 3 times, add trypsin to digest, after the cells become round, add growth medium, resuspend the cells by pipetting, count and control the cell density to 4×10 ⁷ cells/ml.

Select nude mice with a body weight of 18-22 μg, inject 4×10 ⁶ cells into each mouse, and the injection volume is 100 μl/mouse. Five days after cell injection, each mouse was injected once a week with 1 mg of the purified antibody (the activity of the VEGF antibody was equivalent to the activity of 40 μg of the commercial VEGF monoclonal antibody drug avastin), and the purified antibody and the commercialized antibody were respectively used in non-immunized rabbit serum. VEGF monoclonal antibody drug avastin was used as control. The tumor size of the tumor-bearing mice was measured twice a week.

The results are shown in Figure 14, after immunization with VEGF fusion protein vaccine, the antibody can significantly inhibit tumor growth and significantly prolong the survival period of mice (Figure 15).

Comparative example 1

Comparison of VEGF121-CRM197 and VEGF107-CRM197 immunized mice

The methods of Example 2 and Example 3 were used to construct, express and prepare VEGF121-CRM197 recombinant fusion protein and VEGF107-CRM197 recombinant fusion protein respectively. The prepared VEGF121-CRM197 and VEGF107-CRM197 recombinant fusion protein vaccines were used to immunize mice respectively, and the immunization method was as described in Example 4.

Comparing the antibody titers after immunizing mice with VEGF121-CRM197 and VEGF107-CRM197, the results of 1 month after four immunizations are shown in Figure 16. Compared with immunizing mice with VEGF121-CRM197, 14 amino acid residues at the C-terminal were truncated After immunizing mice with VEGF107-CRM197, the titer of anti-VEGF-specific antibodies can be increased by 2-3 times.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A recombinant fusion protein, characterized in that the structure of the fusion protein is as shown in formula I:

   Z1-Z2-Z3 (I)

   Wherein, Z1 is a VEGF antigen fragment element;

   Z2 is a connecting peptide element or none; and

   Z3 is a diphtheria toxin mutant CRM197 protein element;

   "-" indicates a peptide bond or a peptide linker connecting the above elements;

   The amino acid sequence of the VEGF antigen fragment is shown in SEQ ID NO: 5 or SEQ ID NO: 6, which loses VEGF biological activity but retains immunogenicity; and the amino acid sequence of the diphtheria toxin mutant CRM197 protein is shown in SEQ ID NO: Shown in 7.
2. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:12.
3. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO:1.
4. A polynucleotide, characterized in that the polynucleotide encodes the recombinant fusion protein according to claim 1.
5. An expression vector, characterized in that the vector comprises the polynucleotide according to claim 4.
6. A host cell, characterized in that the host cell contains the expression vector according to claim 5 or the polynucleotide according to claim 4 is integrated in the genome.
7. A method for preparing a recombinant fusion protein according to claim 1, comprising the following steps:

   (i) cultivating the host cell as claimed in claim 6;

   (ii) using an inducer to induce the host cell to express the recombinant fusion protein, thereby obtaining the expressed recombinant fusion protein;

   (iii) isolating the expressed recombinant fusion protein, thereby obtaining the isolated recombinant fusion protein.
8. A pharmaceutical composition, characterized in that the composition comprises the recombinant fusion protein of claim 1 and a pharmaceutically acceptable carrier.
9. A recombinant fusion protein as claimed in any one of claims 1-3, a polynucleotide as claimed in claim 4, an expression vector as claimed in claim 5, a host cell as claimed in claim 6 and/or as claimed in claim 8. Use of the pharmaceutical composition in the preparation of medicines for treating and/or preventing diseases.
10. The use according to claim 9, wherein the disease is selected from the group consisting of tumor or cancer, retinal vein occlusion secondary macular edema, wet age-related macular degeneration and diabetic macular edema or a combination thereof.

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